Research ArticleScreening and Molecular Identification of Pectinase ProducingMicrobes from Coffee Pulp

Oliyad Jeilu Oumer 1 and Dawit Abate2

1Department of Biology Ambo University PO Box 19 Ambo Ethiopia2College of Natural Science Addis Ababa University PO Box 1176 Addis Ababa Ethiopia

Correspondence should be addressed to Oliyad Jeilu Oumer oliyadjeiluamboueduet

Received 12 September 2017 Revised 18 January 2018 Accepted 7 February 2018 Published 3 April 2018

Academic Editor Denise Freire

Copyright copy 2018 Oliyad Jeilu Oumer and Dawit Abate This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Application of enzymes in biotechnological process has expanded considerably in recent years In food and related industry majorimportance was being attached to the use of enzymes in upgrading quality increasing yields of extractive processes productstabilization and improvement of flavor and byproduct utilization Pectinases or pectinolytic enzymes are today one of theupcoming enzymes of the commercial sector It has been reported that microbial pectinases account for 25 of the global foodenzymes sales For this reason this study was undertaken with aims of screening microorganisms for the pectinase activity fromcoffee pulp samples and molecular identification of the potential pectinolytic isolates In the present investigation in total ninety-five (95) isolates were identified from thirty coffee pulp samples Based on characterization on the selective growth media theisolates were grouped as actinomycete (2106) bacteria (6526) and fungi (1368) Among these 3158 showed coloniessurrounded by clear zones which indicate the presence of pectinase activity After rigorous screening steps the isolates with highpotential pectinase activity were identified molecularly by sequencing 16S rDNA region of the isolates Based on the molecularidentifications about 70 of the isolates are under genus Bacillus

1 Introduction

Enzymes are natural catalysts They are produced by livingorganisms to increase the rate of an immense and diverseset of chemical reactions required for life They are involvedin all processes essential for life such as DNA replicationand transcription protein synthesis metabolism and signaltransduction Their ability to perform very specific chemicaltransformation has made them increasingly useful in indus-trial processes [1]

In nature microorganisms have been endowed with vastpotentials They produce an array of enzymes which havebeen exploited commercially over the years Todayrsquos enzymetechnology mostly depends on microbes like bacteria andactinomycetes Potential microorganisms are highly suscep-tible to genetic manipulations and hence provide amplescope for strain improvement and for further investigationEcofriendly biotechnological processes seem to be veryimportant as far as themodern society is concerned for which

microbial enzymes are recognized as efficient tools Thus weattempted a study to screen and report enzymes producingmicrobes [2]

The biotechnological potential of pectinolytic enzymesfrom microorganisms has drawn a great deal of attentionfrom various researchers worldwide as likely biological cata-lysts in a variety of industrial processes Pectinolytic enzymescan be applied in various industrial sectors wherever thedegradation of pectin is required for a particular processSeveral microorganisms have been used to produce differ-ent types of pectinolytic enzymes [3] Microbial pectinasesaccount for 25 of the global food and industrial enzymesales [4 5] and theirmarket is increasing day by dayThese areused extensively for fruit juice clarification juice extractionmanufacture of pectin free starch refinement of vegetablefibers degumming of natural fibers wastewater treatmentand cocoa and tobacco and as an analytical tool in the assess-ment of plant products [6 7] Pectinase treatment acceleratestea fermentation and also destroys the foam forming property

HindawiBioMed Research InternationalVolume 2018 Article ID 2961767 7 pageshttpsdoiorg10115520182961767

2 BioMed Research International

of instant tea powders by destroying pectins They are alsoused in coffee fermentation to remove mucilaginous coatfrom coffee beans [8 9]

Although use of pectinases in food-processing industrieshas been fairly well established the modes of action andexperimental utility of several pectin-degrading enzymeshave not been explored for applications in human nutri-tion and health Prebiotics preferred pectic oligosaccharidesduring fermentation Their abilities to protect colonocytesagainst Escherichia coli verocytotoxins to stimulate apoptosisin human colonic adenocarcinoma and to increaseBifidobac-teria and Eubacterium rectale numbers with the subsequentincrease in butyrate concentrations have also been reported[19]

Almost all the commercial preparations of pectinases areproduced from fungal sources Aspergillus niger is the mostcommonly used fungal species for industrial production ofpectinolytic enzymes Thus owing to the enormous poten-tial of pectinase in various sectors of industries wheneverdegradation of pectin is needed it is important to undertakeresearch in screening of microorganisms for pectinase pro-duction

Therefore the present study was conducted with the aimsof screening pectinolytic microorganisms from coffee pulpand identifying molecularly using 16S rRNA

2 Material and Methods

21 Sample Collection A total of 30 coffee pulp samples werecollected from coffee cherry processing site in Gomma Jimazone EthiopiaThe sample collected area is located inOromiaNational Regional State of Ethiopia It is located 390 km southwest of Addis Ababa which is the capital city of EthiopiaThe altitude of this area ranges from 1380 to 1680 metersabove sea level however some points along the southern andwestern boundaries have altitudes ranging from 2229 to 2870meters

About 100 g of each sample was collected aseptically usingUV-rays sterilized polythene bags The sample containingbags were sealed and stored into 4∘C refrigerator inMycologyLaboratory Addis Ababa University until the time of theanalysis

22 Serial Dilution Aseptically 10 g of Coffee Husk fromeach sample was pooled out and homogenized in sterile 90mldistilled water The homogenized samples were agitated for 1hour at 120 rpm on INFORS HT Ecotron incubator shakerand then serially diluted until dilutions 10minus4 and 10minus5

23 Media Preparation Starch casein agar media were pre-pared by dissolving 10 g soluble starch 03 g casein 2 g KNO32 g NaCl 2 g K2HP04 005 g MgSO4sdot7H2O 002 g CaCO3001 g FeSO4sdot7H2O and 15 g agar in 1000ml of distilled waterinside 2 L Erlenmeyer flask The pH of the medium wasadjusted to 70 plusmn 05 using digital pH meter (OAKTON-pH110) The media were sterilized at a temperature of 121∘Cfor 15 minutes Finally about 20ndash25ml of the sterilized starchcasein agar media was poured on sterile Petri plates in

the microbiological hood and allowed to solidify at roomtemperature

Nutrient agar media were prepared by dissolving 28 gof nutrient agar in 1000ml of distilled water inside 2 LErlenmeyer flask The pH of the medium was adjusted to70 plusmn 05 using digital pH meter (OAKTON-pH110) Themedia were sterilized at a temperature of 121∘C for 15minutesFinally about 20ndash25ml of the sterilized nutrient agar mediawas poured on sterile Petri plates in themicrobiological hoodand allowed to solidify at room temperature

Malt extract agar media were prepared by dissolving 50 gofmalt extract agar powder in 1000ml of distilledwater inside2 L Erlenmeyer flask The pH of the medium was adjustedto 70 plusmn 05 using digital pH meter (OAKTON-pH110) Themedia were sterilized at a temperature of 121∘C for 15minutesAfter sterilization themediumallowed to cool up to 50∘Candsupplemented with 1120583gmL of Chloramphenicol to eliminatethe growth of microorganisms other than fungus Finallyabout 20ndash25ml of the sterilized malt extract agar media waspoured on sterile Petri plates in themicrobiological hood andallowed to solidify at room temperature

24 Isolation of Actinomycetes For the isolation of actino-mycetes 01ml aliquots of samples from appropriate dilutionswere inoculated onto sterilized and solidified starch caseinagarmedium using spread platemethod (SCAM) Inoculatedplates were incubated aerobically at 30∘C for 3 daysminus2 weeksin the INFORS HT Ecotron incubator

25 Isolation of Bacteria For the isolation of bacteria 01mlaliquots of samples from appropriate dilutions were inoc-ulated onto sterilized and solidified nutrient agar mediumby spread plate method Inoculated plates were incubatedaerobically at 30∘C for 24ndash48 hours in the INFORS HTEcotron incubator

26 Isolation of Fungi For the isolation of fungi 01ml ali-quots of samples from appropriate dilutions were inoculatedon sterilized and solidified malt extract agar medium byspread plate method Inoculated plates were incubated at30∘C for 3ndash8 days in the INFORS HT Ecotron incubator

27 Purification and Preservation of Cultures Differentcolonies were randomly picked from countable plates (SCAMfor actinomycetes nutrient agar for bacteria and malt extractagar for fungi) and purified by repeated streaking on therespective media Pure cultures of each group of microorgan-isms were then streaked on slants of respective media andstored at 4∘C for further study

28 Presumptive Screening of Isolates for the Pectinase ActivityThe isolates were preliminarily screened for pectinase activityusing pectinase screening agar medium (PSAM) The pH ofthe medium was adjusted to 55 plusmn 05 before sterilization andthen autoclaved with a temperature of 121∘C for 15 minutesFinally 20ndash25ml of media was poured on sterile Petri dishesin the microbiological hood and allowed to solidify at roomtemperature All isolates were streaked into this media andincubated at 30∘C for 24 hours to 2 weeks At the end of

BioMed Research International 3

the incubation period the plates were flooded with 50mMPotassium iodide-iodine solution A clear halo zone aroundthe colonies indicates the ability of an isolate to producepectinase [20]

29 Primary Screening of Efficient Pectinase Producing IsolatesAll the pectinase positive isolates were screened by inoculat-ing them into the above-mentioned screening media Usinga flamed and cooled cork borer a disc of actively growingpectinase positive isolate was taken and transferred to thecenter of screening media and then incubated at 30∘C for24 hours to 2 weeks The ratio of the clear zone diameterto colony diameter during that span of time was measuredin order to select isolates with highest pectinase activityThe largest ratio is assumed to contain the highest activityThose isolates with highest ratio were selected for furtherscreening

210 Secondary Screening of Efficient Pectinase ProducingIsolates Isolates with highest clear zone diameter to colonydiameter ratio in the pectin-agar plates were subjected tosubmerged fermentation using YEP medium The pH of themediumYEPwas adjusted to 70plusmn05 before sterilization andthen autoclaved with a temperature of 121∘C for 15 minutesA volume of 50mL YEP medium in 250mL Erlenmeyerflask was inoculated with 1 inoculum The inoculatedflasks were incubated at 30∘C on an INFORS HT Ecotronincubator shaker at 120 rpm Samples from inoculated flaskswere collected at regular intervals of 24 h and centrifugedat 10000 rpm for 5min at 4∘C The supernatant was usedfor measuring the enzyme activity The enzyme activity wasassayed using sodium acetate buffer pH 65

211 Pectinase Enzyme Assay Pectinase enzyme assay wasbased on the determination of reducing sugars produced asa result of enzymatic hydrolysis of pectin by dinitrosalicylicacid reagent (DNS) method (Miller 1959) For enzyme assay15mL of freshly grown culture was taken and centrifugedat 10000 rpm for 5min The supernatant (100 120583L) from theculture broth was served as the source of the enzyme Inaddition substrate was prepared by mixing 05 (wv) citruspectin in 01M of pH 75 phosphate buffer

From the prepared substrate 900120583l was added to threeclean labeled test tubes one for enzyme one for enzymeblank and one for reagent blank Then 100120583l of crudeenzymewas added to test tube labeled as enzyme and 100 120583l ofdistilled water was added to test tube labeled as regent blankwhile test tube labeled as enzyme blank remained as it wasThen the test tubes were incubated at 50∘C for 10 minutesin the water bath After incubation 2000120583l of dinitrosalicylicacid reagent (DNS) was added to the all test tubes to stopthe reaction Meanwhile into test tube labeled as enzymeblank 100 120583l of crude enzyme was added after the DNSThen all the test tubes were placed in a boiling water bath(92∘C) for 10 minutes Finally the tubes were cooled andoptical density (OD) was measured using spectrophotometer(JENWAY 6300UVVis) at 540 nm Enzyme activity wasmeasured against enzyme blank and reagent blank Theenzyme unit was defined as the amount of enzyme that

catalyzes 120583mol of galacturonic acid per minute (120583molminminus1)under the assay conditions

Relative activity was calculated as the percentage enzymeactivity of the sample with respect to the sample for whichmaximum activity is obtained

Relative Activity =Activity of sample (119880) times 100

Maximum enzyme activity (119880) (1)

212 Molecular Identification The potential isolates whichwere screened and selected on the primary phase wereidentified using molecular techniques

213 Genomic DNA Extraction The potential isolates wereinoculated into 50ml of LB broth (Luria-Bertani) and incu-bated at 37∘C for 24ndash48 hours After incubation they werecentrifuged at 5000 rpm 4∘C for 10min and cells werecollected for genomic DNA extraction The genomic DNAof the isolates was extracted by using the Bacterial GenomicDNA extraction kit according to the manufacturer protocol(QIAGEN QIAamp DNAMini Kit)

214 Quantification and Qualification of DNA by AgaroseGel Electrophoresis The extracted DNA was subjected toagarose gel electrophoresis along with the marker DNA(DNA Ladder) to confirm the quality and quantity About1 g of agarose weighted and poured into 100ml of 1x TAEbuffer to make 1 agarose gel The solution was heated ina microwave oven until agarose gets dissolved The agarosesolution cooled down for about 5 minutes and 05 120583gmL ofethidium bromide (EtBr) added Then the agarose solutionpoured into a gel tray with the well comb in place and itwas allowed to solidify After being solidified the comb wasremoved and the gel was placed in an electrophoretic tankconsisting of 1x TBE buffer About 3 120583l of the extracted DNAwas mixed with 2120583l of the gel loading dye (Bromophenolblue) and it was loaded into the agarose gel wells The gel wasthen electrophoresed at 100 volts for about 45 minutes and itwas observed in a gel documentation system

215 PCR Amplification of the 16S rRNAGene PCR is used toamplify theDNA sequence in between two known sequencesThe amplification of 16s rRNA gene of the isolates was carriedout using PCR machine Two specific primers forwardprimer 27F (51015840-(AGA GTT TGA TCM TGG CTC AG)-31015840)and reverse primer 1492R (51015840-(CGG TTA CCT TGT TACGAC TT)-31015840) which are complementary to the known 16SrDNA sequences were used

216 Setting Up the PCR Reaction and PCRMachine Into theAccuPower Taq PCR PreMix (10 120583l the master mix contain-ing 10x Taq buffer 10mM dNTPs 25mM of MgCl2 1 U ofTaq DNA polymerase) 15 120583l of forward primer (27F) 15 120583lof Reverse primer (1492R) 2 120583l of Genomic DNA and 5 120583lof PCR grade water were added and the PCR amplificationwas done The thermal cycler was programmed for 35 cyclesas follows (1) initial denaturation 94∘C for 2 minutes (2)amplification 94∘C for 45 seconds (denaturation) 56∘C for1 minute (annealing) and 72∘C for 1 minute and 30 seconds

4 BioMed Research International

(extension) (3) final extension 72∘C for 5 minutes and thenheld at 4∘C until needed

217 Quantification and Qualification of Amplified DNA Sam-ples by Nanodrop The amplified DNA might contain impu-rities of RNA and protein that can interfere in the DNAtests About 10 120583l of isolated DNA sample was placed into theNanodrop instrument and quantification and qualification ofamplified DNA was done

218 Gene Sequencing andAnalysis ThePCR sequence prod-ucts were purified and sequenced The obtained sequencedata were compared with known sequences in the GenBankusing the basic local alignment search tool of the nationalcenter for biotechnology information (NCBI) Species wereidentified based on the percentage similarity with the knownspecies sequences in the data base

3 Results

31 Isolation of Microorganisms from Coffee Pulp To isolatemicroorganisms from the collected coffee pulp samples serialdilution pour plating and streak plating isolation techniqueswere used Subsequently the isolates were subcultured intotheir respective selective growth media until pure cultureswere isolated In total ninety-five (95) isolates were identifiedfrom thirty coffee pulp samples Based on characterization onthe selective growth media the isolates were grouped as acti-nomycete (2106) bacteria (6526) and fungi (1368)For identification purpose the isolates were designated byprefix ldquoBtkrdquo and followed by their isolate numbers

32 Screening Isolates for Pectinase Production

321 Presumptive Screening Subsequent to isolation andpurification the isolates were assessed for pectinase activityusing pectinase screening agar medium (PSAM) Amongthe Ninety-five isolates 3158 showed colonies surroundedby clear zones which indicate the presence of pectinaseactivity

322 Primary Screening To identify isolates with higherpectinase activity the ratio between clear zone diameter andcolony diameter was calculated The highest ratio observedwas 47 plusmn 12 by isolate Btk27 Those isolates which scoredhigher than or equal to 20plusmn15 (Meanplusmn SD)were consideredas high enzyme producers which accounts for 333 ofthe pectinase positive isolates (Table 1) These isolates wereselected for molecular identification and further screening

323 Secondary Screening In order to further screen theselected isolates from primary screening submerged fer-mentation test was performed Accordingly subsequent toincubation in YEP media aliquot samples were taken andassayed for their pectinase activity at 65 pH Sodium AcetatebufferThe highest enzyme activities observed were 75plusmn117and 75plusmn052 (Uml) by isolates Btk25 and Btk27 respectively(Table 2) The lowest observed enzyme activity was 002 plusmn001 (Uml) by isolate 42 Btk71

Table 1 Primary screening Isolates clear zone to colony diameterratio

Sr number IsolateClear zone to

colony diameterratiolowast

1 Btk5 29 plusmn 112 Btk23 35 plusmn 183 Btk25 27 plusmn 034 Btk26 34 plusmn 075 Btk27 47 plusmn 126 Btk59 35 plusmn 087 Btk71 27 plusmn 138 Btk73 20 plusmn 159 Btk81 27 plusmn 0310 Btk95 28 plusmn 09lowastValues are mean plusmn SD of triplicates

Table 2 Secondary screening pectinase activity (Unitml)

Sr number Strain number Enzyme activitylowast

1 Btk5 34 plusmn 112b

2 Btk23 66 plusmn 161ba

3 Btk25 75 plusmn 12a

4 Btk26 52 plusmn 05ba

5 Btk27 75 plusmn 05ab

6 Btk59 10 plusmn 04b

7 Btk71 002 plusmn 001b

8 Btk73 31 plusmn 19b

9 Btk81 014 plusmn 001b

10 Btk95 01 plusmn 001b

(i) lowastValues are mean plusmn SD of 3 replicates (ii) Values followed by differentsuperscripts are significantly different at (119875 lt 005) (iii) Values followed bysame superscripts are not significantly different at (119875 lt 005)

33 Molecular Identification of the Isolates The amplifiedPCR products were purified and sequenced The obtainedsequence data were blasted in NCBI database and the likelymicroorganisms (with lower E value higher identity percent-age and maximum total score) were identified (Table 3)

4 Discussion

In recent years the potentials of using microorganisms asbiotechnological sources of industrially relevant enzymeshave stimulated interest in the exploration of extracellularenzymatic activity in several microorganisms [4] Pecti-nase producing microorganisms have advantage over othersources because they can be subjected to genetic andenvironmental manipulations to increase yield [21] In thisstudy potential pectinase producing microorganisms wereisolated using serial dilution pour plating and streak platingtechniques from coffee pulp Similarly [22 23] also followedsimilar procedures to isolate microorganisms from coffeehusk and pulp Coffee pulp is a fibrous mucilaginous materialacquired during the processing of coffee cherries by wet or

BioMed Research International 5

Table 3 Blast (Basic Local Alignment Search Tool) table

Isolate Molecularidentification

GenBank accessionnumber similarity Taxonomy Habitat Reference

Btk5Bacillus

methylotrophicusstrain EGY-SCJ5

KC5734971 97 Bacillus Marine watersediment Egypt [10]

Btk23 Bacillus pumilusstrain B7 KF6418391 95 Bacillus Camel rumen

China [11]

Btk25 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk26Bacillus

amyloliquefaciensstrain ASAG1

FJ597542 95 Bacillus Stored corn China [13]

Btk27 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk59 Exiguobacteriumsp Y11 EF1776901 95 Exiguobacterium Salt Mine China [14]

Btk71Pusillimonas

ginsengisoli strainDCY28

EF6720891 96 Pusillimonas Soil South Korea [15]

Btk73Bacillus

methylotrophicusstrain IHB B 7249

KJ7673541 97 BacillusCamellia sinensis

(tea)India

[16]

Btk81 Bacillus spSVUNM JX1192401 74 Bacillus Mica mines India [17]

Btk95 Staphylococcus spNR7 EU7848441 88 Staphylococcus Sausage China [18]

dry process respectively The presence of proteins pectinother sugars and minerals and its high humidity favor therapid growth of pectinolytic microorganisms Moreover theorganic nature of the material makes it an ideal substratefor microbial processes for the production of value-addedproducts [24]

Emerging new applications underline the importance ofscreening pectinase producing microorganisms with novelproperties greater enzyme activity and large-scale pro-duction of these enzymes [25] In this study the isolateswere subjected into plate agar and submerged fermentationscreening methods to identify potent isolate with highestenzyme activity and enzyme activity with broad pH rangesThe study is in agreement with [26] reporting plate agarscreening method used to screen native isolates for pectinaseactivity Mehta et al [27] screened bacterial strains isolatedfrom soil and samples of vegetable using plate agar andsubmerged fermentation screening methods

Pectinases are a heterogeneous group of related enzymesand according to the cleavage site they are divided intothree groups (1) polygalacturonase (2) pectin lyase andpectate lyase (3) pectin esterase [28 29] Polygalacturonases(PGases) have been reported in many microorganismsincluding Neurospora crassa Aspergillus sp and Bacillussp [5 30] Pectin esterase is reported in Pseudomonassolanacearum Aspergillus niger Lactobacillus lactis Penicil-lium occitanis and A japonicus[4 5 31] Pectate lyases areproduced by many bacteria and some pathogenic fungiThey have been reported in Erwinia carotovora Pseudomonas

syringae and Bacillus sp [5 32] Pectin lyases have beenreported to be produced by Aspergillus japonicus Penicilliumsp and Aspergillus sp [5 30 33] In this study pectinaseactivity is determined on the basis of measuring the amountof reducing sugar by colorimetric methods specifically using5-dinitrosalicylate reagent method Based on the assay pro-cedures and characteristics of the pectinase the pectinase ofthe screened and identified isolates in this study resemblespolygalacturonase

The potential isolates for pectinase production wereidentified molecularly using the 16S rRNA gene Amongthe molecularly identified isolates about 70 of the isolateswere under genus of Bacillus According to Priest (1977)there was a widespread distribution of pectinolytic activitythroughout the genus of Bacillus Some works also had beendone to produce pectinase by many strains of these genus[32 34ndash37] In addition during screening the isolates whichsecrete the highest quantity of pectinase was also of genusBacillus The result is in good agreement with Namasivayamet al [34] which reported that Bacillus sp can produce largequantities of extracellular pectinase enzyme Also out ofthe ten molecularly identified isolates six out of them havepercentage similarity on the database lt97 therefore thoseisolates might be a novel or a new strainspecies [38]

Disclosure

This article does not contain any studies with human partici-pants or animals performed by any of the authors

6 BioMed Research International

Conflicts of Interest

There are no conflicts of interest between the authors

Acknowledgments

The authors are delighted to acknowledge Addis AbabaUniversity andAmboUniversity for their cooperation duringthis study

References

[1] S Li X Yang S Yang M Zhu and X Wang ldquoTechnologyprospecting on enzymes application marketing and engineer-ingrdquo Computational and Structural Biotechnology Journal vol2 no 3 p e201209017 2012

[2] S Vuppu and M Mishra ldquoAn Overview of Some ReportedSoil Enzyme Producing Microorganismsrdquo Indian Journal ofFundamental and Applied Life Sciences vol 1 no 4 pp 180-862011

[3] R S Jayani S K Shukla and R Gupta ldquoScreening of bacterialstrains for polygalacturonase activity its production by bacillussphaericus (MTCC 7542)rdquo Enzyme Research vol 2010 ArticleID 306785 5 pages 2010

[4] R S Jayani S Saxena and R Gupta ldquoMicrobial pectinolyticenzymes a reviewrdquo Process Biochemistry vol 40 no 9 pp 2931ndash2944 2005

[5] H A Murad and H H Azzaz ldquoMicrobial pectinases andruminant nutritionrdquoResearch Journal ofMicrobiology vol 6 no3 pp 246ndash269 2011

[6] I Alkorta C Garbisu M J Llama and J L Serra ldquoIndustrialapplications of pectic enzymes a reviewrdquo Process Biochemistryvol 33 no 1 pp 21ndash28 1998

[7] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[8] C Sieiro B Garcıa-Fraga J Lopez-Seijas A F Da Silva andT G Villa ldquoMicrobial Pectic Enzymes in the Food and WineIndustry Food Ind Process - Methods Equiprdquo 2012 httpwwwintechopencombooksfood-industrial-processes-meth-ods-and-equipmentmicrobial-pectic-enzymes-in-the-food-and-wine-industry

[9] G Hoondal R Tiwari R Tewari N Dahiya and Q BegldquoMicrobial alkaline pectinases and their industrial applicationsA reviewrdquo Applied Microbiology and Biotechnology vol 59 no4-5 pp 409ndash418 2002

[10] R A Amer F Mapelli H M El Gendi et al ldquoBacterial diver-sity and bioremediation potential of the highly contaminatedmarine sediments at elmax district (Egyptmediterranean sea)rdquoBioMed Research International vol 2015 17 pages 2015

[11] J Meerak H Iida Y Watanabe et al ldquoPhylogeny of 120574-polyglutamic acid-producing Bacillus strains isolated fromfermented soybean foods manufactured in Asian countriesrdquoThe Journal of General and Applied Microbiology vol 53 no 6pp 315ndash323 2007

[12] J Yue L Liu andZChen Institution of Plant Protection JiangsuAcademy Agriculture Nanjing Jiangsu 210014 China 2010

[13] Y Lin D Du C Si Q Zhao Z Li and P Li ldquoPotentialbiocontrol Bacillus sp strains isolated by an improved methodfrom vinegar waste compost exhibit antibiosis against fungal

pathogens and promote growth of cucumbersrdquo Biological Con-trol vol 71 pp 7ndash15 2014

[14] Y G Chen H M Li Q Y Li et al Phylogenetic Diversity ofCulturable Bacteria in the Ancient Salt Deposits of the YipinglangSalt Mine P R China 2006

[15] N Lodders and P Kampfer ldquoA combined cultivation andcultivation-independent approach shows high bacterial diver-sity in water-miscible metalworking fluidsrdquo Systematic andApplied Microbiology vol 35 no 4 pp 246ndash252 2012

[16] A Gulati S Sood and R Thakur Plant Pathology andMicrobiology Lab HATS Division CSIR-Institute of HimalayanBioresource Technology Post Box No 6 Palampur HimachalPradesh 176061 India 2016

[17] E Li C M Hamm A S Gulati et al ldquoInflammatory bowel dis-eases phenotype C difficile and NOD2 genotype are associatedwith shifts in human ileum associated microbial compositionrdquoPLoS ONE vol 7 no 6 Article ID e26284 2012

[18] H H Kong J Oh C Deming et al ldquoTemporal shifts in theskin microbiome associated with disease flares and treatmentin children with atopic dermatitisrdquo Genome Research vol 22no 5 pp 850ndash859 2012

[19] M Khan E Nakkeeran and S Umesh-Kumar ldquoPotentialapplication of pectinase in developing functional foodsrdquoAnnualReview of Food Science and Technology vol 4 no 1 pp 21ndash342013

[20] Q K Beg B Bhushan M Kapoor and G S Hoondal ldquoPro-duction and characterization of thermostable xylanase andpectinase from Streptomyces sp QG-11-3rdquo Journal of IndustrialMicrobiology and Biotechnology vol 24 no 6 pp 396ndash4022000

[21] V Bhardwaj and G Neelam ldquoProduction Purification of Pecti-nase from Bacillus Sp MBRL576 Isolate and Its Applicationin Extraction of Juicerdquo International Journal of Science andResearch vol 3 no 6 pp 648ndash652 2014

[22] S Nayak M J Harshitha M Maithili C Sampath H SAnilkumar and C Vaman Rao ldquoIsolation and characterizationof caffeine degrading bacteria from coffee pulprdquo Indian Journalof Biochemistry and Biophysics vol 11 no 1 pp 86ndash91 2012

[23] J Sumitha and T Sivakumar ldquoDevelopment Centrerdquo in Orig-inal Research Article Isolation and Characterization of CaffeineDegrading Bacteria from West Karnataka India N Tamil Edvol 2 pp 338ndash346 2013

[24] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[25] S Shruti and K Sudev Mandal ldquoOptimization of Process-ing Parameters for Production of Pectinolytic Enzymes fromFermented Pineapple Residue of Mixed Aspergillus SpeciesrdquoJordan Journal of Biological Sciences vol 5 no 4 pp 307ndash3142012

[26] P Hitha K and D Girija ldquoIsolation and Screening of NativeMicrobial Isolates for Pectinase Activityrdquo International Journalof Science and Research vol 3 no 5 pp 632ndash634 2014

[27] S AMehta RMitali S Nilofer and P Nimisha ldquoOptimizationof Physiological Parameters for Pectinase Production From SoilIsolates and Its Applications in Fruit Juice Clarificationrdquo Journalof Environmental Research And Development vol 7 no 4 pp1539ndash1546 2013

[28] P K Yadav V K Singh S Yadav K D S Yadav and D YadavldquoIn silico analysis of pectin lyase and pectinase sequencesrdquoBiochemistry (Moscow) vol 74 no 9 pp 1049ndash1055 2009

BioMed Research International 3

the incubation period the plates were flooded with 50mMPotassium iodide-iodine solution A clear halo zone aroundthe colonies indicates the ability of an isolate to producepectinase [20]

29 Primary Screening of Efficient Pectinase Producing IsolatesAll the pectinase positive isolates were screened by inoculat-ing them into the above-mentioned screening media Usinga flamed and cooled cork borer a disc of actively growingpectinase positive isolate was taken and transferred to thecenter of screening media and then incubated at 30∘C for24 hours to 2 weeks The ratio of the clear zone diameterto colony diameter during that span of time was measuredin order to select isolates with highest pectinase activityThe largest ratio is assumed to contain the highest activityThose isolates with highest ratio were selected for furtherscreening

210 Secondary Screening of Efficient Pectinase ProducingIsolates Isolates with highest clear zone diameter to colonydiameter ratio in the pectin-agar plates were subjected tosubmerged fermentation using YEP medium The pH of themediumYEPwas adjusted to 70plusmn05 before sterilization andthen autoclaved with a temperature of 121∘C for 15 minutesA volume of 50mL YEP medium in 250mL Erlenmeyerflask was inoculated with 1 inoculum The inoculatedflasks were incubated at 30∘C on an INFORS HT Ecotronincubator shaker at 120 rpm Samples from inoculated flaskswere collected at regular intervals of 24 h and centrifugedat 10000 rpm for 5min at 4∘C The supernatant was usedfor measuring the enzyme activity The enzyme activity wasassayed using sodium acetate buffer pH 65

211 Pectinase Enzyme Assay Pectinase enzyme assay wasbased on the determination of reducing sugars produced asa result of enzymatic hydrolysis of pectin by dinitrosalicylicacid reagent (DNS) method (Miller 1959) For enzyme assay15mL of freshly grown culture was taken and centrifugedat 10000 rpm for 5min The supernatant (100 120583L) from theculture broth was served as the source of the enzyme Inaddition substrate was prepared by mixing 05 (wv) citruspectin in 01M of pH 75 phosphate buffer

From the prepared substrate 900120583l was added to threeclean labeled test tubes one for enzyme one for enzymeblank and one for reagent blank Then 100120583l of crudeenzymewas added to test tube labeled as enzyme and 100 120583l ofdistilled water was added to test tube labeled as regent blankwhile test tube labeled as enzyme blank remained as it wasThen the test tubes were incubated at 50∘C for 10 minutesin the water bath After incubation 2000120583l of dinitrosalicylicacid reagent (DNS) was added to the all test tubes to stopthe reaction Meanwhile into test tube labeled as enzymeblank 100 120583l of crude enzyme was added after the DNSThen all the test tubes were placed in a boiling water bath(92∘C) for 10 minutes Finally the tubes were cooled andoptical density (OD) was measured using spectrophotometer(JENWAY 6300UVVis) at 540 nm Enzyme activity wasmeasured against enzyme blank and reagent blank Theenzyme unit was defined as the amount of enzyme that

catalyzes 120583mol of galacturonic acid per minute (120583molminminus1)under the assay conditions

Relative activity was calculated as the percentage enzymeactivity of the sample with respect to the sample for whichmaximum activity is obtained

Relative Activity =Activity of sample (119880) times 100

Maximum enzyme activity (119880) (1)

212 Molecular Identification The potential isolates whichwere screened and selected on the primary phase wereidentified using molecular techniques

213 Genomic DNA Extraction The potential isolates wereinoculated into 50ml of LB broth (Luria-Bertani) and incu-bated at 37∘C for 24ndash48 hours After incubation they werecentrifuged at 5000 rpm 4∘C for 10min and cells werecollected for genomic DNA extraction The genomic DNAof the isolates was extracted by using the Bacterial GenomicDNA extraction kit according to the manufacturer protocol(QIAGEN QIAamp DNAMini Kit)

214 Quantification and Qualification of DNA by AgaroseGel Electrophoresis The extracted DNA was subjected toagarose gel electrophoresis along with the marker DNA(DNA Ladder) to confirm the quality and quantity About1 g of agarose weighted and poured into 100ml of 1x TAEbuffer to make 1 agarose gel The solution was heated ina microwave oven until agarose gets dissolved The agarosesolution cooled down for about 5 minutes and 05 120583gmL ofethidium bromide (EtBr) added Then the agarose solutionpoured into a gel tray with the well comb in place and itwas allowed to solidify After being solidified the comb wasremoved and the gel was placed in an electrophoretic tankconsisting of 1x TBE buffer About 3 120583l of the extracted DNAwas mixed with 2120583l of the gel loading dye (Bromophenolblue) and it was loaded into the agarose gel wells The gel wasthen electrophoresed at 100 volts for about 45 minutes and itwas observed in a gel documentation system

215 PCR Amplification of the 16S rRNAGene PCR is used toamplify theDNA sequence in between two known sequencesThe amplification of 16s rRNA gene of the isolates was carriedout using PCR machine Two specific primers forwardprimer 27F (51015840-(AGA GTT TGA TCM TGG CTC AG)-31015840)and reverse primer 1492R (51015840-(CGG TTA CCT TGT TACGAC TT)-31015840) which are complementary to the known 16SrDNA sequences were used

216 Setting Up the PCR Reaction and PCRMachine Into theAccuPower Taq PCR PreMix (10 120583l the master mix contain-ing 10x Taq buffer 10mM dNTPs 25mM of MgCl2 1 U ofTaq DNA polymerase) 15 120583l of forward primer (27F) 15 120583lof Reverse primer (1492R) 2 120583l of Genomic DNA and 5 120583lof PCR grade water were added and the PCR amplificationwas done The thermal cycler was programmed for 35 cyclesas follows (1) initial denaturation 94∘C for 2 minutes (2)amplification 94∘C for 45 seconds (denaturation) 56∘C for1 minute (annealing) and 72∘C for 1 minute and 30 seconds

4 BioMed Research International

(extension) (3) final extension 72∘C for 5 minutes and thenheld at 4∘C until needed

217 Quantification and Qualification of Amplified DNA Sam-ples by Nanodrop The amplified DNA might contain impu-rities of RNA and protein that can interfere in the DNAtests About 10 120583l of isolated DNA sample was placed into theNanodrop instrument and quantification and qualification ofamplified DNA was done

218 Gene Sequencing andAnalysis ThePCR sequence prod-ucts were purified and sequenced The obtained sequencedata were compared with known sequences in the GenBankusing the basic local alignment search tool of the nationalcenter for biotechnology information (NCBI) Species wereidentified based on the percentage similarity with the knownspecies sequences in the data base

3 Results

31 Isolation of Microorganisms from Coffee Pulp To isolatemicroorganisms from the collected coffee pulp samples serialdilution pour plating and streak plating isolation techniqueswere used Subsequently the isolates were subcultured intotheir respective selective growth media until pure cultureswere isolated In total ninety-five (95) isolates were identifiedfrom thirty coffee pulp samples Based on characterization onthe selective growth media the isolates were grouped as acti-nomycete (2106) bacteria (6526) and fungi (1368)For identification purpose the isolates were designated byprefix ldquoBtkrdquo and followed by their isolate numbers

32 Screening Isolates for Pectinase Production

321 Presumptive Screening Subsequent to isolation andpurification the isolates were assessed for pectinase activityusing pectinase screening agar medium (PSAM) Amongthe Ninety-five isolates 3158 showed colonies surroundedby clear zones which indicate the presence of pectinaseactivity

322 Primary Screening To identify isolates with higherpectinase activity the ratio between clear zone diameter andcolony diameter was calculated The highest ratio observedwas 47 plusmn 12 by isolate Btk27 Those isolates which scoredhigher than or equal to 20plusmn15 (Meanplusmn SD)were consideredas high enzyme producers which accounts for 333 ofthe pectinase positive isolates (Table 1) These isolates wereselected for molecular identification and further screening

323 Secondary Screening In order to further screen theselected isolates from primary screening submerged fer-mentation test was performed Accordingly subsequent toincubation in YEP media aliquot samples were taken andassayed for their pectinase activity at 65 pH Sodium AcetatebufferThe highest enzyme activities observed were 75plusmn117and 75plusmn052 (Uml) by isolates Btk25 and Btk27 respectively(Table 2) The lowest observed enzyme activity was 002 plusmn001 (Uml) by isolate 42 Btk71

Table 1 Primary screening Isolates clear zone to colony diameterratio

Sr number IsolateClear zone to

colony diameterratiolowast

1 Btk5 29 plusmn 112 Btk23 35 plusmn 183 Btk25 27 plusmn 034 Btk26 34 plusmn 075 Btk27 47 plusmn 126 Btk59 35 plusmn 087 Btk71 27 plusmn 138 Btk73 20 plusmn 159 Btk81 27 plusmn 0310 Btk95 28 plusmn 09lowastValues are mean plusmn SD of triplicates

Table 2 Secondary screening pectinase activity (Unitml)

Sr number Strain number Enzyme activitylowast

1 Btk5 34 plusmn 112b

2 Btk23 66 plusmn 161ba

3 Btk25 75 plusmn 12a

4 Btk26 52 plusmn 05ba

5 Btk27 75 plusmn 05ab

6 Btk59 10 plusmn 04b

7 Btk71 002 plusmn 001b

8 Btk73 31 plusmn 19b

9 Btk81 014 plusmn 001b

10 Btk95 01 plusmn 001b

(i) lowastValues are mean plusmn SD of 3 replicates (ii) Values followed by differentsuperscripts are significantly different at (119875 lt 005) (iii) Values followed bysame superscripts are not significantly different at (119875 lt 005)

33 Molecular Identification of the Isolates The amplifiedPCR products were purified and sequenced The obtainedsequence data were blasted in NCBI database and the likelymicroorganisms (with lower E value higher identity percent-age and maximum total score) were identified (Table 3)

4 Discussion

In recent years the potentials of using microorganisms asbiotechnological sources of industrially relevant enzymeshave stimulated interest in the exploration of extracellularenzymatic activity in several microorganisms [4] Pecti-nase producing microorganisms have advantage over othersources because they can be subjected to genetic andenvironmental manipulations to increase yield [21] In thisstudy potential pectinase producing microorganisms wereisolated using serial dilution pour plating and streak platingtechniques from coffee pulp Similarly [22 23] also followedsimilar procedures to isolate microorganisms from coffeehusk and pulp Coffee pulp is a fibrous mucilaginous materialacquired during the processing of coffee cherries by wet or

BioMed Research International 5

Table 3 Blast (Basic Local Alignment Search Tool) table

Isolate Molecularidentification

GenBank accessionnumber similarity Taxonomy Habitat Reference

Btk5Bacillus

methylotrophicusstrain EGY-SCJ5

KC5734971 97 Bacillus Marine watersediment Egypt [10]

Btk23 Bacillus pumilusstrain B7 KF6418391 95 Bacillus Camel rumen

China [11]

Btk25 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk26Bacillus

amyloliquefaciensstrain ASAG1

FJ597542 95 Bacillus Stored corn China [13]

Btk27 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk59 Exiguobacteriumsp Y11 EF1776901 95 Exiguobacterium Salt Mine China [14]

Btk71Pusillimonas

ginsengisoli strainDCY28

EF6720891 96 Pusillimonas Soil South Korea [15]

Btk73Bacillus

methylotrophicusstrain IHB B 7249

KJ7673541 97 BacillusCamellia sinensis

(tea)India

[16]

Btk81 Bacillus spSVUNM JX1192401 74 Bacillus Mica mines India [17]

Btk95 Staphylococcus spNR7 EU7848441 88 Staphylococcus Sausage China [18]

dry process respectively The presence of proteins pectinother sugars and minerals and its high humidity favor therapid growth of pectinolytic microorganisms Moreover theorganic nature of the material makes it an ideal substratefor microbial processes for the production of value-addedproducts [24]

Emerging new applications underline the importance ofscreening pectinase producing microorganisms with novelproperties greater enzyme activity and large-scale pro-duction of these enzymes [25] In this study the isolateswere subjected into plate agar and submerged fermentationscreening methods to identify potent isolate with highestenzyme activity and enzyme activity with broad pH rangesThe study is in agreement with [26] reporting plate agarscreening method used to screen native isolates for pectinaseactivity Mehta et al [27] screened bacterial strains isolatedfrom soil and samples of vegetable using plate agar andsubmerged fermentation screening methods

Pectinases are a heterogeneous group of related enzymesand according to the cleavage site they are divided intothree groups (1) polygalacturonase (2) pectin lyase andpectate lyase (3) pectin esterase [28 29] Polygalacturonases(PGases) have been reported in many microorganismsincluding Neurospora crassa Aspergillus sp and Bacillussp [5 30] Pectin esterase is reported in Pseudomonassolanacearum Aspergillus niger Lactobacillus lactis Penicil-lium occitanis and A japonicus[4 5 31] Pectate lyases areproduced by many bacteria and some pathogenic fungiThey have been reported in Erwinia carotovora Pseudomonas

syringae and Bacillus sp [5 32] Pectin lyases have beenreported to be produced by Aspergillus japonicus Penicilliumsp and Aspergillus sp [5 30 33] In this study pectinaseactivity is determined on the basis of measuring the amountof reducing sugar by colorimetric methods specifically using5-dinitrosalicylate reagent method Based on the assay pro-cedures and characteristics of the pectinase the pectinase ofthe screened and identified isolates in this study resemblespolygalacturonase

The potential isolates for pectinase production wereidentified molecularly using the 16S rRNA gene Amongthe molecularly identified isolates about 70 of the isolateswere under genus of Bacillus According to Priest (1977)there was a widespread distribution of pectinolytic activitythroughout the genus of Bacillus Some works also had beendone to produce pectinase by many strains of these genus[32 34ndash37] In addition during screening the isolates whichsecrete the highest quantity of pectinase was also of genusBacillus The result is in good agreement with Namasivayamet al [34] which reported that Bacillus sp can produce largequantities of extracellular pectinase enzyme Also out ofthe ten molecularly identified isolates six out of them havepercentage similarity on the database lt97 therefore thoseisolates might be a novel or a new strainspecies [38]

Disclosure

This article does not contain any studies with human partici-pants or animals performed by any of the authors

6 BioMed Research International

Conflicts of Interest

There are no conflicts of interest between the authors

Acknowledgments

The authors are delighted to acknowledge Addis AbabaUniversity andAmboUniversity for their cooperation duringthis study

References

[1] S Li X Yang S Yang M Zhu and X Wang ldquoTechnologyprospecting on enzymes application marketing and engineer-ingrdquo Computational and Structural Biotechnology Journal vol2 no 3 p e201209017 2012

[2] S Vuppu and M Mishra ldquoAn Overview of Some ReportedSoil Enzyme Producing Microorganismsrdquo Indian Journal ofFundamental and Applied Life Sciences vol 1 no 4 pp 180-862011

[3] R S Jayani S K Shukla and R Gupta ldquoScreening of bacterialstrains for polygalacturonase activity its production by bacillussphaericus (MTCC 7542)rdquo Enzyme Research vol 2010 ArticleID 306785 5 pages 2010

[4] R S Jayani S Saxena and R Gupta ldquoMicrobial pectinolyticenzymes a reviewrdquo Process Biochemistry vol 40 no 9 pp 2931ndash2944 2005

[5] H A Murad and H H Azzaz ldquoMicrobial pectinases andruminant nutritionrdquoResearch Journal ofMicrobiology vol 6 no3 pp 246ndash269 2011

[6] I Alkorta C Garbisu M J Llama and J L Serra ldquoIndustrialapplications of pectic enzymes a reviewrdquo Process Biochemistryvol 33 no 1 pp 21ndash28 1998

[7] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[8] C Sieiro B Garcıa-Fraga J Lopez-Seijas A F Da Silva andT G Villa ldquoMicrobial Pectic Enzymes in the Food and WineIndustry Food Ind Process - Methods Equiprdquo 2012 httpwwwintechopencombooksfood-industrial-processes-meth-ods-and-equipmentmicrobial-pectic-enzymes-in-the-food-and-wine-industry

[9] G Hoondal R Tiwari R Tewari N Dahiya and Q BegldquoMicrobial alkaline pectinases and their industrial applicationsA reviewrdquo Applied Microbiology and Biotechnology vol 59 no4-5 pp 409ndash418 2002

[10] R A Amer F Mapelli H M El Gendi et al ldquoBacterial diver-sity and bioremediation potential of the highly contaminatedmarine sediments at elmax district (Egyptmediterranean sea)rdquoBioMed Research International vol 2015 17 pages 2015

[11] J Meerak H Iida Y Watanabe et al ldquoPhylogeny of 120574-polyglutamic acid-producing Bacillus strains isolated fromfermented soybean foods manufactured in Asian countriesrdquoThe Journal of General and Applied Microbiology vol 53 no 6pp 315ndash323 2007

[12] J Yue L Liu andZChen Institution of Plant Protection JiangsuAcademy Agriculture Nanjing Jiangsu 210014 China 2010

[13] Y Lin D Du C Si Q Zhao Z Li and P Li ldquoPotentialbiocontrol Bacillus sp strains isolated by an improved methodfrom vinegar waste compost exhibit antibiosis against fungal

pathogens and promote growth of cucumbersrdquo Biological Con-trol vol 71 pp 7ndash15 2014

[14] Y G Chen H M Li Q Y Li et al Phylogenetic Diversity ofCulturable Bacteria in the Ancient Salt Deposits of the YipinglangSalt Mine P R China 2006

[15] N Lodders and P Kampfer ldquoA combined cultivation andcultivation-independent approach shows high bacterial diver-sity in water-miscible metalworking fluidsrdquo Systematic andApplied Microbiology vol 35 no 4 pp 246ndash252 2012

[16] A Gulati S Sood and R Thakur Plant Pathology andMicrobiology Lab HATS Division CSIR-Institute of HimalayanBioresource Technology Post Box No 6 Palampur HimachalPradesh 176061 India 2016

[17] E Li C M Hamm A S Gulati et al ldquoInflammatory bowel dis-eases phenotype C difficile and NOD2 genotype are associatedwith shifts in human ileum associated microbial compositionrdquoPLoS ONE vol 7 no 6 Article ID e26284 2012

[18] H H Kong J Oh C Deming et al ldquoTemporal shifts in theskin microbiome associated with disease flares and treatmentin children with atopic dermatitisrdquo Genome Research vol 22no 5 pp 850ndash859 2012

[19] M Khan E Nakkeeran and S Umesh-Kumar ldquoPotentialapplication of pectinase in developing functional foodsrdquoAnnualReview of Food Science and Technology vol 4 no 1 pp 21ndash342013

[20] Q K Beg B Bhushan M Kapoor and G S Hoondal ldquoPro-duction and characterization of thermostable xylanase andpectinase from Streptomyces sp QG-11-3rdquo Journal of IndustrialMicrobiology and Biotechnology vol 24 no 6 pp 396ndash4022000

[21] V Bhardwaj and G Neelam ldquoProduction Purification of Pecti-nase from Bacillus Sp MBRL576 Isolate and Its Applicationin Extraction of Juicerdquo International Journal of Science andResearch vol 3 no 6 pp 648ndash652 2014

[22] S Nayak M J Harshitha M Maithili C Sampath H SAnilkumar and C Vaman Rao ldquoIsolation and characterizationof caffeine degrading bacteria from coffee pulprdquo Indian Journalof Biochemistry and Biophysics vol 11 no 1 pp 86ndash91 2012

[23] J Sumitha and T Sivakumar ldquoDevelopment Centrerdquo in Orig-inal Research Article Isolation and Characterization of CaffeineDegrading Bacteria from West Karnataka India N Tamil Edvol 2 pp 338ndash346 2013

[24] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[25] S Shruti and K Sudev Mandal ldquoOptimization of Process-ing Parameters for Production of Pectinolytic Enzymes fromFermented Pineapple Residue of Mixed Aspergillus SpeciesrdquoJordan Journal of Biological Sciences vol 5 no 4 pp 307ndash3142012

[26] P Hitha K and D Girija ldquoIsolation and Screening of NativeMicrobial Isolates for Pectinase Activityrdquo International Journalof Science and Research vol 3 no 5 pp 632ndash634 2014

[27] S AMehta RMitali S Nilofer and P Nimisha ldquoOptimizationof Physiological Parameters for Pectinase Production From SoilIsolates and Its Applications in Fruit Juice Clarificationrdquo Journalof Environmental Research And Development vol 7 no 4 pp1539ndash1546 2013

[28] P K Yadav V K Singh S Yadav K D S Yadav and D YadavldquoIn silico analysis of pectin lyase and pectinase sequencesrdquoBiochemistry (Moscow) vol 74 no 9 pp 1049ndash1055 2009

4 BioMed Research International

(extension) (3) final extension 72∘C for 5 minutes and thenheld at 4∘C until needed

217 Quantification and Qualification of Amplified DNA Sam-ples by Nanodrop The amplified DNA might contain impu-rities of RNA and protein that can interfere in the DNAtests About 10 120583l of isolated DNA sample was placed into theNanodrop instrument and quantification and qualification ofamplified DNA was done

218 Gene Sequencing andAnalysis ThePCR sequence prod-ucts were purified and sequenced The obtained sequencedata were compared with known sequences in the GenBankusing the basic local alignment search tool of the nationalcenter for biotechnology information (NCBI) Species wereidentified based on the percentage similarity with the knownspecies sequences in the data base

3 Results

31 Isolation of Microorganisms from Coffee Pulp To isolatemicroorganisms from the collected coffee pulp samples serialdilution pour plating and streak plating isolation techniqueswere used Subsequently the isolates were subcultured intotheir respective selective growth media until pure cultureswere isolated In total ninety-five (95) isolates were identifiedfrom thirty coffee pulp samples Based on characterization onthe selective growth media the isolates were grouped as acti-nomycete (2106) bacteria (6526) and fungi (1368)For identification purpose the isolates were designated byprefix ldquoBtkrdquo and followed by their isolate numbers

32 Screening Isolates for Pectinase Production

321 Presumptive Screening Subsequent to isolation andpurification the isolates were assessed for pectinase activityusing pectinase screening agar medium (PSAM) Amongthe Ninety-five isolates 3158 showed colonies surroundedby clear zones which indicate the presence of pectinaseactivity

322 Primary Screening To identify isolates with higherpectinase activity the ratio between clear zone diameter andcolony diameter was calculated The highest ratio observedwas 47 plusmn 12 by isolate Btk27 Those isolates which scoredhigher than or equal to 20plusmn15 (Meanplusmn SD)were consideredas high enzyme producers which accounts for 333 ofthe pectinase positive isolates (Table 1) These isolates wereselected for molecular identification and further screening

323 Secondary Screening In order to further screen theselected isolates from primary screening submerged fer-mentation test was performed Accordingly subsequent toincubation in YEP media aliquot samples were taken andassayed for their pectinase activity at 65 pH Sodium AcetatebufferThe highest enzyme activities observed were 75plusmn117and 75plusmn052 (Uml) by isolates Btk25 and Btk27 respectively(Table 2) The lowest observed enzyme activity was 002 plusmn001 (Uml) by isolate 42 Btk71

Table 1 Primary screening Isolates clear zone to colony diameterratio

Sr number IsolateClear zone to

colony diameterratiolowast

1 Btk5 29 plusmn 112 Btk23 35 plusmn 183 Btk25 27 plusmn 034 Btk26 34 plusmn 075 Btk27 47 plusmn 126 Btk59 35 plusmn 087 Btk71 27 plusmn 138 Btk73 20 plusmn 159 Btk81 27 plusmn 0310 Btk95 28 plusmn 09lowastValues are mean plusmn SD of triplicates

Table 2 Secondary screening pectinase activity (Unitml)

Sr number Strain number Enzyme activitylowast

1 Btk5 34 plusmn 112b

2 Btk23 66 plusmn 161ba

3 Btk25 75 plusmn 12a

4 Btk26 52 plusmn 05ba

5 Btk27 75 plusmn 05ab

6 Btk59 10 plusmn 04b

7 Btk71 002 plusmn 001b

8 Btk73 31 plusmn 19b

9 Btk81 014 plusmn 001b

10 Btk95 01 plusmn 001b

(i) lowastValues are mean plusmn SD of 3 replicates (ii) Values followed by differentsuperscripts are significantly different at (119875 lt 005) (iii) Values followed bysame superscripts are not significantly different at (119875 lt 005)

33 Molecular Identification of the Isolates The amplifiedPCR products were purified and sequenced The obtainedsequence data were blasted in NCBI database and the likelymicroorganisms (with lower E value higher identity percent-age and maximum total score) were identified (Table 3)

4 Discussion

In recent years the potentials of using microorganisms asbiotechnological sources of industrially relevant enzymeshave stimulated interest in the exploration of extracellularenzymatic activity in several microorganisms [4] Pecti-nase producing microorganisms have advantage over othersources because they can be subjected to genetic andenvironmental manipulations to increase yield [21] In thisstudy potential pectinase producing microorganisms wereisolated using serial dilution pour plating and streak platingtechniques from coffee pulp Similarly [22 23] also followedsimilar procedures to isolate microorganisms from coffeehusk and pulp Coffee pulp is a fibrous mucilaginous materialacquired during the processing of coffee cherries by wet or

BioMed Research International 5

Table 3 Blast (Basic Local Alignment Search Tool) table

Isolate Molecularidentification

GenBank accessionnumber similarity Taxonomy Habitat Reference

Btk5Bacillus

methylotrophicusstrain EGY-SCJ5

KC5734971 97 Bacillus Marine watersediment Egypt [10]

Btk23 Bacillus pumilusstrain B7 KF6418391 95 Bacillus Camel rumen

China [11]

Btk25 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk26Bacillus

amyloliquefaciensstrain ASAG1

FJ597542 95 Bacillus Stored corn China [13]

Btk27 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk59 Exiguobacteriumsp Y11 EF1776901 95 Exiguobacterium Salt Mine China [14]

Btk71Pusillimonas

ginsengisoli strainDCY28

EF6720891 96 Pusillimonas Soil South Korea [15]

Btk73Bacillus

methylotrophicusstrain IHB B 7249

KJ7673541 97 BacillusCamellia sinensis

(tea)India

[16]

Btk81 Bacillus spSVUNM JX1192401 74 Bacillus Mica mines India [17]

Btk95 Staphylococcus spNR7 EU7848441 88 Staphylococcus Sausage China [18]

dry process respectively The presence of proteins pectinother sugars and minerals and its high humidity favor therapid growth of pectinolytic microorganisms Moreover theorganic nature of the material makes it an ideal substratefor microbial processes for the production of value-addedproducts [24]

Emerging new applications underline the importance ofscreening pectinase producing microorganisms with novelproperties greater enzyme activity and large-scale pro-duction of these enzymes [25] In this study the isolateswere subjected into plate agar and submerged fermentationscreening methods to identify potent isolate with highestenzyme activity and enzyme activity with broad pH rangesThe study is in agreement with [26] reporting plate agarscreening method used to screen native isolates for pectinaseactivity Mehta et al [27] screened bacterial strains isolatedfrom soil and samples of vegetable using plate agar andsubmerged fermentation screening methods

Pectinases are a heterogeneous group of related enzymesand according to the cleavage site they are divided intothree groups (1) polygalacturonase (2) pectin lyase andpectate lyase (3) pectin esterase [28 29] Polygalacturonases(PGases) have been reported in many microorganismsincluding Neurospora crassa Aspergillus sp and Bacillussp [5 30] Pectin esterase is reported in Pseudomonassolanacearum Aspergillus niger Lactobacillus lactis Penicil-lium occitanis and A japonicus[4 5 31] Pectate lyases areproduced by many bacteria and some pathogenic fungiThey have been reported in Erwinia carotovora Pseudomonas

syringae and Bacillus sp [5 32] Pectin lyases have beenreported to be produced by Aspergillus japonicus Penicilliumsp and Aspergillus sp [5 30 33] In this study pectinaseactivity is determined on the basis of measuring the amountof reducing sugar by colorimetric methods specifically using5-dinitrosalicylate reagent method Based on the assay pro-cedures and characteristics of the pectinase the pectinase ofthe screened and identified isolates in this study resemblespolygalacturonase

The potential isolates for pectinase production wereidentified molecularly using the 16S rRNA gene Amongthe molecularly identified isolates about 70 of the isolateswere under genus of Bacillus According to Priest (1977)there was a widespread distribution of pectinolytic activitythroughout the genus of Bacillus Some works also had beendone to produce pectinase by many strains of these genus[32 34ndash37] In addition during screening the isolates whichsecrete the highest quantity of pectinase was also of genusBacillus The result is in good agreement with Namasivayamet al [34] which reported that Bacillus sp can produce largequantities of extracellular pectinase enzyme Also out ofthe ten molecularly identified isolates six out of them havepercentage similarity on the database lt97 therefore thoseisolates might be a novel or a new strainspecies [38]

Disclosure

This article does not contain any studies with human partici-pants or animals performed by any of the authors

6 BioMed Research International

Conflicts of Interest

There are no conflicts of interest between the authors

Acknowledgments

The authors are delighted to acknowledge Addis AbabaUniversity andAmboUniversity for their cooperation duringthis study

References

[1] S Li X Yang S Yang M Zhu and X Wang ldquoTechnologyprospecting on enzymes application marketing and engineer-ingrdquo Computational and Structural Biotechnology Journal vol2 no 3 p e201209017 2012

[2] S Vuppu and M Mishra ldquoAn Overview of Some ReportedSoil Enzyme Producing Microorganismsrdquo Indian Journal ofFundamental and Applied Life Sciences vol 1 no 4 pp 180-862011

[3] R S Jayani S K Shukla and R Gupta ldquoScreening of bacterialstrains for polygalacturonase activity its production by bacillussphaericus (MTCC 7542)rdquo Enzyme Research vol 2010 ArticleID 306785 5 pages 2010

[4] R S Jayani S Saxena and R Gupta ldquoMicrobial pectinolyticenzymes a reviewrdquo Process Biochemistry vol 40 no 9 pp 2931ndash2944 2005

[5] H A Murad and H H Azzaz ldquoMicrobial pectinases andruminant nutritionrdquoResearch Journal ofMicrobiology vol 6 no3 pp 246ndash269 2011

[6] I Alkorta C Garbisu M J Llama and J L Serra ldquoIndustrialapplications of pectic enzymes a reviewrdquo Process Biochemistryvol 33 no 1 pp 21ndash28 1998

[7] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[8] C Sieiro B Garcıa-Fraga J Lopez-Seijas A F Da Silva andT G Villa ldquoMicrobial Pectic Enzymes in the Food and WineIndustry Food Ind Process - Methods Equiprdquo 2012 httpwwwintechopencombooksfood-industrial-processes-meth-ods-and-equipmentmicrobial-pectic-enzymes-in-the-food-and-wine-industry

[9] G Hoondal R Tiwari R Tewari N Dahiya and Q BegldquoMicrobial alkaline pectinases and their industrial applicationsA reviewrdquo Applied Microbiology and Biotechnology vol 59 no4-5 pp 409ndash418 2002

[10] R A Amer F Mapelli H M El Gendi et al ldquoBacterial diver-sity and bioremediation potential of the highly contaminatedmarine sediments at elmax district (Egyptmediterranean sea)rdquoBioMed Research International vol 2015 17 pages 2015

[11] J Meerak H Iida Y Watanabe et al ldquoPhylogeny of 120574-polyglutamic acid-producing Bacillus strains isolated fromfermented soybean foods manufactured in Asian countriesrdquoThe Journal of General and Applied Microbiology vol 53 no 6pp 315ndash323 2007

[12] J Yue L Liu andZChen Institution of Plant Protection JiangsuAcademy Agriculture Nanjing Jiangsu 210014 China 2010

[13] Y Lin D Du C Si Q Zhao Z Li and P Li ldquoPotentialbiocontrol Bacillus sp strains isolated by an improved methodfrom vinegar waste compost exhibit antibiosis against fungal

pathogens and promote growth of cucumbersrdquo Biological Con-trol vol 71 pp 7ndash15 2014

[14] Y G Chen H M Li Q Y Li et al Phylogenetic Diversity ofCulturable Bacteria in the Ancient Salt Deposits of the YipinglangSalt Mine P R China 2006

[15] N Lodders and P Kampfer ldquoA combined cultivation andcultivation-independent approach shows high bacterial diver-sity in water-miscible metalworking fluidsrdquo Systematic andApplied Microbiology vol 35 no 4 pp 246ndash252 2012

[16] A Gulati S Sood and R Thakur Plant Pathology andMicrobiology Lab HATS Division CSIR-Institute of HimalayanBioresource Technology Post Box No 6 Palampur HimachalPradesh 176061 India 2016

[17] E Li C M Hamm A S Gulati et al ldquoInflammatory bowel dis-eases phenotype C difficile and NOD2 genotype are associatedwith shifts in human ileum associated microbial compositionrdquoPLoS ONE vol 7 no 6 Article ID e26284 2012

[18] H H Kong J Oh C Deming et al ldquoTemporal shifts in theskin microbiome associated with disease flares and treatmentin children with atopic dermatitisrdquo Genome Research vol 22no 5 pp 850ndash859 2012

[19] M Khan E Nakkeeran and S Umesh-Kumar ldquoPotentialapplication of pectinase in developing functional foodsrdquoAnnualReview of Food Science and Technology vol 4 no 1 pp 21ndash342013

[20] Q K Beg B Bhushan M Kapoor and G S Hoondal ldquoPro-duction and characterization of thermostable xylanase andpectinase from Streptomyces sp QG-11-3rdquo Journal of IndustrialMicrobiology and Biotechnology vol 24 no 6 pp 396ndash4022000

[21] V Bhardwaj and G Neelam ldquoProduction Purification of Pecti-nase from Bacillus Sp MBRL576 Isolate and Its Applicationin Extraction of Juicerdquo International Journal of Science andResearch vol 3 no 6 pp 648ndash652 2014

[22] S Nayak M J Harshitha M Maithili C Sampath H SAnilkumar and C Vaman Rao ldquoIsolation and characterizationof caffeine degrading bacteria from coffee pulprdquo Indian Journalof Biochemistry and Biophysics vol 11 no 1 pp 86ndash91 2012

[23] J Sumitha and T Sivakumar ldquoDevelopment Centrerdquo in Orig-inal Research Article Isolation and Characterization of CaffeineDegrading Bacteria from West Karnataka India N Tamil Edvol 2 pp 338ndash346 2013

[24] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[25] S Shruti and K Sudev Mandal ldquoOptimization of Process-ing Parameters for Production of Pectinolytic Enzymes fromFermented Pineapple Residue of Mixed Aspergillus SpeciesrdquoJordan Journal of Biological Sciences vol 5 no 4 pp 307ndash3142012

[26] P Hitha K and D Girija ldquoIsolation and Screening of NativeMicrobial Isolates for Pectinase Activityrdquo International Journalof Science and Research vol 3 no 5 pp 632ndash634 2014

[27] S AMehta RMitali S Nilofer and P Nimisha ldquoOptimizationof Physiological Parameters for Pectinase Production From SoilIsolates and Its Applications in Fruit Juice Clarificationrdquo Journalof Environmental Research And Development vol 7 no 4 pp1539ndash1546 2013

[28] P K Yadav V K Singh S Yadav K D S Yadav and D YadavldquoIn silico analysis of pectin lyase and pectinase sequencesrdquoBiochemistry (Moscow) vol 74 no 9 pp 1049ndash1055 2009

BioMed Research International 5

Table 3 Blast (Basic Local Alignment Search Tool) table

Isolate Molecularidentification

GenBank accessionnumber similarity Taxonomy Habitat Reference

Btk5Bacillus

methylotrophicusstrain EGY-SCJ5

KC5734971 97 Bacillus Marine watersediment Egypt [10]

Btk23 Bacillus pumilusstrain B7 KF6418391 95 Bacillus Camel rumen

China [11]

Btk25 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk26Bacillus

amyloliquefaciensstrain ASAG1

FJ597542 95 Bacillus Stored corn China [13]

Btk27 Bacillus subtilisstrain NBT-15 HQ2445011 97 Bacillus Penut China [12]

Btk59 Exiguobacteriumsp Y11 EF1776901 95 Exiguobacterium Salt Mine China [14]

Btk71Pusillimonas

ginsengisoli strainDCY28

EF6720891 96 Pusillimonas Soil South Korea [15]

Btk73Bacillus

methylotrophicusstrain IHB B 7249

KJ7673541 97 BacillusCamellia sinensis

(tea)India

[16]

Btk81 Bacillus spSVUNM JX1192401 74 Bacillus Mica mines India [17]

Btk95 Staphylococcus spNR7 EU7848441 88 Staphylococcus Sausage China [18]

dry process respectively The presence of proteins pectinother sugars and minerals and its high humidity favor therapid growth of pectinolytic microorganisms Moreover theorganic nature of the material makes it an ideal substratefor microbial processes for the production of value-addedproducts [24]

Emerging new applications underline the importance ofscreening pectinase producing microorganisms with novelproperties greater enzyme activity and large-scale pro-duction of these enzymes [25] In this study the isolateswere subjected into plate agar and submerged fermentationscreening methods to identify potent isolate with highestenzyme activity and enzyme activity with broad pH rangesThe study is in agreement with [26] reporting plate agarscreening method used to screen native isolates for pectinaseactivity Mehta et al [27] screened bacterial strains isolatedfrom soil and samples of vegetable using plate agar andsubmerged fermentation screening methods

Pectinases are a heterogeneous group of related enzymesand according to the cleavage site they are divided intothree groups (1) polygalacturonase (2) pectin lyase andpectate lyase (3) pectin esterase [28 29] Polygalacturonases(PGases) have been reported in many microorganismsincluding Neurospora crassa Aspergillus sp and Bacillussp [5 30] Pectin esterase is reported in Pseudomonassolanacearum Aspergillus niger Lactobacillus lactis Penicil-lium occitanis and A japonicus[4 5 31] Pectate lyases areproduced by many bacteria and some pathogenic fungiThey have been reported in Erwinia carotovora Pseudomonas

syringae and Bacillus sp [5 32] Pectin lyases have beenreported to be produced by Aspergillus japonicus Penicilliumsp and Aspergillus sp [5 30 33] In this study pectinaseactivity is determined on the basis of measuring the amountof reducing sugar by colorimetric methods specifically using5-dinitrosalicylate reagent method Based on the assay pro-cedures and characteristics of the pectinase the pectinase ofthe screened and identified isolates in this study resemblespolygalacturonase

The potential isolates for pectinase production wereidentified molecularly using the 16S rRNA gene Amongthe molecularly identified isolates about 70 of the isolateswere under genus of Bacillus According to Priest (1977)there was a widespread distribution of pectinolytic activitythroughout the genus of Bacillus Some works also had beendone to produce pectinase by many strains of these genus[32 34ndash37] In addition during screening the isolates whichsecrete the highest quantity of pectinase was also of genusBacillus The result is in good agreement with Namasivayamet al [34] which reported that Bacillus sp can produce largequantities of extracellular pectinase enzyme Also out ofthe ten molecularly identified isolates six out of them havepercentage similarity on the database lt97 therefore thoseisolates might be a novel or a new strainspecies [38]

Disclosure

This article does not contain any studies with human partici-pants or animals performed by any of the authors

6 BioMed Research International

Conflicts of Interest

There are no conflicts of interest between the authors

Acknowledgments

The authors are delighted to acknowledge Addis AbabaUniversity andAmboUniversity for their cooperation duringthis study

References

[1] S Li X Yang S Yang M Zhu and X Wang ldquoTechnologyprospecting on enzymes application marketing and engineer-ingrdquo Computational and Structural Biotechnology Journal vol2 no 3 p e201209017 2012

[2] S Vuppu and M Mishra ldquoAn Overview of Some ReportedSoil Enzyme Producing Microorganismsrdquo Indian Journal ofFundamental and Applied Life Sciences vol 1 no 4 pp 180-862011

[3] R S Jayani S K Shukla and R Gupta ldquoScreening of bacterialstrains for polygalacturonase activity its production by bacillussphaericus (MTCC 7542)rdquo Enzyme Research vol 2010 ArticleID 306785 5 pages 2010

[4] R S Jayani S Saxena and R Gupta ldquoMicrobial pectinolyticenzymes a reviewrdquo Process Biochemistry vol 40 no 9 pp 2931ndash2944 2005

[5] H A Murad and H H Azzaz ldquoMicrobial pectinases andruminant nutritionrdquoResearch Journal ofMicrobiology vol 6 no3 pp 246ndash269 2011

[6] I Alkorta C Garbisu M J Llama and J L Serra ldquoIndustrialapplications of pectic enzymes a reviewrdquo Process Biochemistryvol 33 no 1 pp 21ndash28 1998

[7] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[8] C Sieiro B Garcıa-Fraga J Lopez-Seijas A F Da Silva andT G Villa ldquoMicrobial Pectic Enzymes in the Food and WineIndustry Food Ind Process - Methods Equiprdquo 2012 httpwwwintechopencombooksfood-industrial-processes-meth-ods-and-equipmentmicrobial-pectic-enzymes-in-the-food-and-wine-industry

[9] G Hoondal R Tiwari R Tewari N Dahiya and Q BegldquoMicrobial alkaline pectinases and their industrial applicationsA reviewrdquo Applied Microbiology and Biotechnology vol 59 no4-5 pp 409ndash418 2002

[10] R A Amer F Mapelli H M El Gendi et al ldquoBacterial diver-sity and bioremediation potential of the highly contaminatedmarine sediments at elmax district (Egyptmediterranean sea)rdquoBioMed Research International vol 2015 17 pages 2015

[11] J Meerak H Iida Y Watanabe et al ldquoPhylogeny of 120574-polyglutamic acid-producing Bacillus strains isolated fromfermented soybean foods manufactured in Asian countriesrdquoThe Journal of General and Applied Microbiology vol 53 no 6pp 315ndash323 2007

[12] J Yue L Liu andZChen Institution of Plant Protection JiangsuAcademy Agriculture Nanjing Jiangsu 210014 China 2010

[13] Y Lin D Du C Si Q Zhao Z Li and P Li ldquoPotentialbiocontrol Bacillus sp strains isolated by an improved methodfrom vinegar waste compost exhibit antibiosis against fungal

pathogens and promote growth of cucumbersrdquo Biological Con-trol vol 71 pp 7ndash15 2014

[14] Y G Chen H M Li Q Y Li et al Phylogenetic Diversity ofCulturable Bacteria in the Ancient Salt Deposits of the YipinglangSalt Mine P R China 2006

[15] N Lodders and P Kampfer ldquoA combined cultivation andcultivation-independent approach shows high bacterial diver-sity in water-miscible metalworking fluidsrdquo Systematic andApplied Microbiology vol 35 no 4 pp 246ndash252 2012

[16] A Gulati S Sood and R Thakur Plant Pathology andMicrobiology Lab HATS Division CSIR-Institute of HimalayanBioresource Technology Post Box No 6 Palampur HimachalPradesh 176061 India 2016

[17] E Li C M Hamm A S Gulati et al ldquoInflammatory bowel dis-eases phenotype C difficile and NOD2 genotype are associatedwith shifts in human ileum associated microbial compositionrdquoPLoS ONE vol 7 no 6 Article ID e26284 2012

[18] H H Kong J Oh C Deming et al ldquoTemporal shifts in theskin microbiome associated with disease flares and treatmentin children with atopic dermatitisrdquo Genome Research vol 22no 5 pp 850ndash859 2012

[19] M Khan E Nakkeeran and S Umesh-Kumar ldquoPotentialapplication of pectinase in developing functional foodsrdquoAnnualReview of Food Science and Technology vol 4 no 1 pp 21ndash342013

[20] Q K Beg B Bhushan M Kapoor and G S Hoondal ldquoPro-duction and characterization of thermostable xylanase andpectinase from Streptomyces sp QG-11-3rdquo Journal of IndustrialMicrobiology and Biotechnology vol 24 no 6 pp 396ndash4022000

[21] V Bhardwaj and G Neelam ldquoProduction Purification of Pecti-nase from Bacillus Sp MBRL576 Isolate and Its Applicationin Extraction of Juicerdquo International Journal of Science andResearch vol 3 no 6 pp 648ndash652 2014

[22] S Nayak M J Harshitha M Maithili C Sampath H SAnilkumar and C Vaman Rao ldquoIsolation and characterizationof caffeine degrading bacteria from coffee pulprdquo Indian Journalof Biochemistry and Biophysics vol 11 no 1 pp 86ndash91 2012

[23] J Sumitha and T Sivakumar ldquoDevelopment Centrerdquo in Orig-inal Research Article Isolation and Characterization of CaffeineDegrading Bacteria from West Karnataka India N Tamil Edvol 2 pp 338ndash346 2013

[24] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[25] S Shruti and K Sudev Mandal ldquoOptimization of Process-ing Parameters for Production of Pectinolytic Enzymes fromFermented Pineapple Residue of Mixed Aspergillus SpeciesrdquoJordan Journal of Biological Sciences vol 5 no 4 pp 307ndash3142012

[26] P Hitha K and D Girija ldquoIsolation and Screening of NativeMicrobial Isolates for Pectinase Activityrdquo International Journalof Science and Research vol 3 no 5 pp 632ndash634 2014

[27] S AMehta RMitali S Nilofer and P Nimisha ldquoOptimizationof Physiological Parameters for Pectinase Production From SoilIsolates and Its Applications in Fruit Juice Clarificationrdquo Journalof Environmental Research And Development vol 7 no 4 pp1539ndash1546 2013

[28] P K Yadav V K Singh S Yadav K D S Yadav and D YadavldquoIn silico analysis of pectin lyase and pectinase sequencesrdquoBiochemistry (Moscow) vol 74 no 9 pp 1049ndash1055 2009

6 BioMed Research International

Conflicts of Interest

There are no conflicts of interest between the authors

Acknowledgments

The authors are delighted to acknowledge Addis AbabaUniversity andAmboUniversity for their cooperation duringthis study

References

[1] S Li X Yang S Yang M Zhu and X Wang ldquoTechnologyprospecting on enzymes application marketing and engineer-ingrdquo Computational and Structural Biotechnology Journal vol2 no 3 p e201209017 2012

[2] S Vuppu and M Mishra ldquoAn Overview of Some ReportedSoil Enzyme Producing Microorganismsrdquo Indian Journal ofFundamental and Applied Life Sciences vol 1 no 4 pp 180-862011

[3] R S Jayani S K Shukla and R Gupta ldquoScreening of bacterialstrains for polygalacturonase activity its production by bacillussphaericus (MTCC 7542)rdquo Enzyme Research vol 2010 ArticleID 306785 5 pages 2010

[4] R S Jayani S Saxena and R Gupta ldquoMicrobial pectinolyticenzymes a reviewrdquo Process Biochemistry vol 40 no 9 pp 2931ndash2944 2005

[5] H A Murad and H H Azzaz ldquoMicrobial pectinases andruminant nutritionrdquoResearch Journal ofMicrobiology vol 6 no3 pp 246ndash269 2011

[6] I Alkorta C Garbisu M J Llama and J L Serra ldquoIndustrialapplications of pectic enzymes a reviewrdquo Process Biochemistryvol 33 no 1 pp 21ndash28 1998

[7] S A Singh M Ramakrishna and A G Appu Rao ldquoOptimisa-tion of downstream processing parameters for the recovery ofpectinase from the fermented bran of Aspergillus carbonariusrdquoProcess Biochemistry vol 35 no 3-4 pp 411ndash417 1999

[8] C Sieiro B Garcıa-Fraga J Lopez-Seijas A F Da Silva andT G Villa ldquoMicrobial Pectic Enzymes in the Food and WineIndustry Food Ind Process - Methods Equiprdquo 2012 httpwwwintechopencombooksfood-industrial-processes-meth-ods-and-equipmentmicrobial-pectic-enzymes-in-the-food-and-wine-industry

[9] G Hoondal R Tiwari R Tewari N Dahiya and Q BegldquoMicrobial alkaline pectinases and their industrial applicationsA reviewrdquo Applied Microbiology and Biotechnology vol 59 no4-5 pp 409ndash418 2002

[10] R A Amer F Mapelli H M El Gendi et al ldquoBacterial diver-sity and bioremediation potential of the highly contaminatedmarine sediments at elmax district (Egyptmediterranean sea)rdquoBioMed Research International vol 2015 17 pages 2015

[11] J Meerak H Iida Y Watanabe et al ldquoPhylogeny of 120574-polyglutamic acid-producing Bacillus strains isolated fromfermented soybean foods manufactured in Asian countriesrdquoThe Journal of General and Applied Microbiology vol 53 no 6pp 315ndash323 2007

[12] J Yue L Liu andZChen Institution of Plant Protection JiangsuAcademy Agriculture Nanjing Jiangsu 210014 China 2010

[13] Y Lin D Du C Si Q Zhao Z Li and P Li ldquoPotentialbiocontrol Bacillus sp strains isolated by an improved methodfrom vinegar waste compost exhibit antibiosis against fungal

pathogens and promote growth of cucumbersrdquo Biological Con-trol vol 71 pp 7ndash15 2014

[14] Y G Chen H M Li Q Y Li et al Phylogenetic Diversity ofCulturable Bacteria in the Ancient Salt Deposits of the YipinglangSalt Mine P R China 2006

[15] N Lodders and P Kampfer ldquoA combined cultivation andcultivation-independent approach shows high bacterial diver-sity in water-miscible metalworking fluidsrdquo Systematic andApplied Microbiology vol 35 no 4 pp 246ndash252 2012

[16] A Gulati S Sood and R Thakur Plant Pathology andMicrobiology Lab HATS Division CSIR-Institute of HimalayanBioresource Technology Post Box No 6 Palampur HimachalPradesh 176061 India 2016

[17] E Li C M Hamm A S Gulati et al ldquoInflammatory bowel dis-eases phenotype C difficile and NOD2 genotype are associatedwith shifts in human ileum associated microbial compositionrdquoPLoS ONE vol 7 no 6 Article ID e26284 2012

[18] H H Kong J Oh C Deming et al ldquoTemporal shifts in theskin microbiome associated with disease flares and treatmentin children with atopic dermatitisrdquo Genome Research vol 22no 5 pp 850ndash859 2012

[19] M Khan E Nakkeeran and S Umesh-Kumar ldquoPotentialapplication of pectinase in developing functional foodsrdquoAnnualReview of Food Science and Technology vol 4 no 1 pp 21ndash342013

[20] Q K Beg B Bhushan M Kapoor and G S Hoondal ldquoPro-duction and characterization of thermostable xylanase andpectinase from Streptomyces sp QG-11-3rdquo Journal of IndustrialMicrobiology and Biotechnology vol 24 no 6 pp 396ndash4022000

[21] V Bhardwaj and G Neelam ldquoProduction Purification of Pecti-nase from Bacillus Sp MBRL576 Isolate and Its Applicationin Extraction of Juicerdquo International Journal of Science andResearch vol 3 no 6 pp 648ndash652 2014

[22] S Nayak M J Harshitha M Maithili C Sampath H SAnilkumar and C Vaman Rao ldquoIsolation and characterizationof caffeine degrading bacteria from coffee pulprdquo Indian Journalof Biochemistry and Biophysics vol 11 no 1 pp 86ndash91 2012

[23] J Sumitha and T Sivakumar ldquoDevelopment Centrerdquo in Orig-inal Research Article Isolation and Characterization of CaffeineDegrading Bacteria from West Karnataka India N Tamil Edvol 2 pp 338ndash346 2013

[24] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[25] S Shruti and K Sudev Mandal ldquoOptimization of Process-ing Parameters for Production of Pectinolytic Enzymes fromFermented Pineapple Residue of Mixed Aspergillus SpeciesrdquoJordan Journal of Biological Sciences vol 5 no 4 pp 307ndash3142012

[26] P Hitha K and D Girija ldquoIsolation and Screening of NativeMicrobial Isolates for Pectinase Activityrdquo International Journalof Science and Research vol 3 no 5 pp 632ndash634 2014

[27] S AMehta RMitali S Nilofer and P Nimisha ldquoOptimizationof Physiological Parameters for Pectinase Production From SoilIsolates and Its Applications in Fruit Juice Clarificationrdquo Journalof Environmental Research And Development vol 7 no 4 pp1539ndash1546 2013

[28] P K Yadav V K Singh S Yadav K D S Yadav and D YadavldquoIn silico analysis of pectin lyase and pectinase sequencesrdquoBiochemistry (Moscow) vol 74 no 9 pp 1049ndash1055 2009

BioMed Research International 7

[29] D J Osborne ldquoAdvances in Pectin and Pectinase Researchrdquo inAnnals of Botany F Voragen H Schols and R Visser Eds vol94 pp 479-480TheNetherlands KluwerAcademic Publishers2004

[30] D R Kashyap P K Vohra S Chopra and R Tewari ldquoAppli-cations of pectinases in the commercial sector a reviewrdquoBioresource Technology vol 77 no 3 pp 215ndash227 2001

[31] C Arunachalam and S Asha ldquoPectinolytic Enzyme - A Reviewof New Studiesrdquo Adv Biotech J vol 561 pp 1ndash5 2010

[32] D R Kashyap S Chandra A Kaul and R Tewari ldquoProductionpurification and characterization of pectinase fromaBacillus spDT7rdquoWorld Journal of Microbiology and Biotechnology vol 16no 3 pp 277ndash282 2000

[33] A Chaudhri and V Suneetha ldquoMicrobially Derived PectinasesA Reviewrdquo IOSR Journal of Pharmacy and Biological Sciencesvol 2 no 2 pp 1ndash5 2012

[34] E Namasivayam D John Ravindar K Mariappan A jiji MKumar and R L Jayaraj ldquoProduction of extracellular pectinaseby bacillus cereus isolated from market solid wasterdquo Journal ofBioanalysis amp Biomedicine vol 3 no 3 pp 70ndash75 2011

[35] R M El-Shishtawy S A Mohamed A M Asiri A-B MGomaa I H Ibrahim and H A Al-Talhi ldquoSolid fermentationof wheat bran for hydrolytic enzymes production and sacchar-ification content by a local isolate Bacillus megatheriumrdquo BMCBiotechnology vol 14 article no 29 2014

[36] H Yuanyuan L Sixin H Xiong and L Congfa ldquoOptimiza-tion of Polygalacturonase Production Condition for BacillusLicheniformis 3 x 05 Using Response Surfacerdquo Int Conf AgricBiosyst Eng Adv Biomed Eng pp 373ndash377 2011

[37] A Roosdiana S Prasetyawan C Mahdi and S SutrisnoldquoProduction and Characterization of Bacillus firmus pectinaserdquoThe Journal of Pure and Applied Chemistry Research vol 2 no1 pp 35ndash41 2013

[38] P Yarza P Yilmaz E Pruesse et al ldquoUniting the classification ofcultured and uncultured bacteria and archaea using 16S rRNAgene sequencesrdquoNature Reviews Microbiology vol 12 no 9 pp635ndash645 2014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom