MINI-REVIEW

Nocardiopsis species as potential sources of diverse and novelextracellular enzymes

Tahsin Bennur & Ameeta Ravi Kumar & Smita Zinjarde &

Vaishali Javdekar

Received: 1 July 2014 /Revised: 18 September 2014 /Accepted: 19 September 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Members of the genus Nocardiopsis are generallyencountered in locations that are inherently extreme. They arepresent in frozen soils, desert sand, compost, saline or hyper-saline habitats (marine systems, salterns and soils) and alka-line places (slag dumps, lake soils and sediments). In order tosurvive under these severe conditions, they produce novel anddiverse enzymes that allow them to utilize the available nutri-ents and to thrive. The members of this genus are multifacetedand release an assortment of extracellular hydrolytic enzymes.They produce enzymes that are cold-adapted (α-amylases),t h e rmo t o l e r a n t (α - amy l a s e s a n d xy l a n a s e s ) ,thermoalkalotolerant (cellulases, β-1,3-glucanases), alkali-tolerant thermostable (inulinases), acid-stable (keratinase)and alkalophilic (serine proteases). Some of the enzymesderived from Nocardiopsis species act on insoluble polymerssuch as glucans (pachyman and curdlan), keratin (feathers andprion proteins) and polyhydroxyalkanoates. Extreme toler-ance exhibited by proteases has been attributed to the presenceof some amino acids (Asn and Pro) in loop structures, reloca-tion of multiple salt bridges to outer regions of the protein orthe presence of a distinct polyproline II helix. The range ofnovel enzymes is projected to increase in the forthcomingyears, as new isolates are being continually reported, and thedevelopment of processes involving such enzymes is envis-aged in the future.

Keywords Nocardiopsis . Carbohydrases .

PHB depolymerases . Proteases

Introduction

Actinomycetes are a major group of Gram-positive filamen-tous bacteria with high guanine and cytosine contents. Theyare undoubtedly most significant with respect to synthesis ofantibiotics and other bioactive compounds. However, theyalso produce a variety of extracellular hydrolytic enzymes thatmediate degradation and recycling of natural biopolymers(Olano et al. 2009). Several actinomycetes, includingNocardiopsis species, are isolated from different sites, andthey inherently have a vast range of enzymatic abilities.Meena et al. (2013) have studied actinobacteria from a tropicalmarine hot spot (Andaman and Nicobar Islands) and havedescribed them as potential sources for industrial andpharmaceutical products. Genera such as Saccharopolyspora,Streptomyces, Streptoverticillium, Microtetraspora,Actinopolyspora, Actinokineospora, Dactylosporangium andNocardiopsis were obtained during this study. A majority ofthese isolates produced industrially important enzymes such asamylases, proteases, gelatinases, lipases, DNases, cellulases,ureases and phosphatases. On another occasion, Kumar et al.(2012) have isolated actinomycetes from earthworm castingsand have screened them for antimicrobial activities andindustrial enzymes. Streptomyces, Streptosporangium,Saccharopolyspora , Nocardia , Micromonospora ,Actinomadura, Microbispora, Planobispora and Nocardiopsiswere obtained. Several of these isolates produced amylases,caseinases, cellulases, gelatinases, xylanases and lipases. Tanet al. (2009) have screened the actinomycetes associated withgoat faeces for their hydrolytic enzymes. Oerskovia,Streptomyces and Nocardiopsis species were the importantgenera that were obtained, and they produced enzymes such

T. Bennur :A. R. Kumar : S. Zinjarde (*)Institute of Bioinformatics and Biotechnology, University of Pune,Pune 411007, Indiae-mail: smita@unipune.ac.in

V. Javdekar (*)Department of Biotechnology, Abasaheb Garware College,Pune 411004, Indiae-mail: vaishali.javdekar@mesagc.org

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-6111-y

as amylases, proteases and galactosidases. On the basis of thesereports, it is evident that members of the genus Nocardiopsisalong with other actinomycetes are present in diverse environ-ments, and they are important enzyme producers.

Species of Nocardiopsis are strictly aerobic, Gram-posi-tive, non-acid-fast actinomycetes (Grund and Kroppenstedt1990). From the taxonomic point of view, members of thegenus Nocardiopsis belong to phylum Actinobacteria, classActinobacteria, order Actinomycetales and familyNocardiopsaceae (Rainey et al. 1996; Sun et al. 2010). A recentreview indicates that there are 42 species included in this genus(Hamedi et al. 2013). In the years to come, this count isexpected to increase. The organism is endowed with avariety of unusual physiological and metabolic features,and a large number of research laboratories have beenusing this as a model system for understanding funda-mental phenomena related to enzymes and in developingapplications as discussed in the following sections.

Nocardiopsis species are intrinsically present in a vastrange of ecological habitats. There are reports on the isolationof this actinomycete from a variety of harsh environments assummarized in Fig. 1. With respect to extreme temperatures,some species have been isolated from cold soils (Xu et al.2014), and others have been obtained from desert sand dunesand compost soils, where temperatures are higher (Hozzeinand Goodfellow 2008; Yamamura et al. 2010; Yan et al.2011). A large plethora of species has been isolated fromenvironments that are rich in salt. In this regard, Hamediet al. (2013) in a recent review article have describedNocardiopsis species as the most abundant halophilic andhalotolerant actinomycete. Among the 42 hitherto reportedspecies, 13 are halophilic and six are halotolerant. They havebeen isolated frommarine sediments (Fang et al. 2011; Meenaet al. 2013), hypersaline solar salterns (Chun et al. 2000; Joseand Jebakumar 2012), saline soils (Al-Tai and Ruan 1994; Liet al. 2003; Li et al. 2006; Chen et al. 2010) and marinebiological forms (Chen et al. 2009; Li et al. 2012). In addition,some species are alkali-tolerant being inherently present in

alkali slag dumps, lake soils and sediments (Schippers et al.2002; Yang et al. 2008; Mwirichia et al. 2010). Li et al. (2013)on the basis of comparative genomic analysis have concludedthat the high versatility and adaptability of Nocardiopsis spe-cies are due to their intrinsic genetic features.

Since the members of genus Nocardiopsis are widespread inenvironmentally harsh conditions, it was deduced that the en-zymes produced by this actinomycete would also be diverse andnovel. It must be noted that extremophiles are of considerablesignificance with respect to (i) understanding structure-functionrelations of their constituent enzymes, (ii) studying the adaptabil-ity of such organisms to extreme environmental conditions and(iii) providing enzymes that can be used as advanced tools for avariety of applications (Niehaus et al. 1999; Vieille and Zeikus2001; de Champdore et al. 2007; Gabani and Singh 2013;Elleuche et al. 2014). A thorough assessment of the availableliterature on the topic led to this exclusive review highlighting theproduction, biochemical properties, biophysical characterizationand applications of the extracellular enzymes produced by thisorganism. To the best of our knowledge, there are no recentreviews on the enzymes produced by this biotechnologicallyrelevant genus. In this review, current updates on the enzymesproduced by this actinomycete have been broadly categorized onthe basis of their substrates and on the type of environments fromwhich the different strains producing these diverse enzymes havebeen isolated.

Carbohydrases obtained from Nocardiopsis species

A range of carbohydrases is produced by Nocardiopsis species.Starch, cellulose, β-glucans, inulin, xylan and chitin are naturalbiopolymers that are degraded by extracellular amylases (EC3.2.1.1), cellulases (EC 3.2.1.4), β-1,3-glucanases (EC3.2.1.39), inulinases (EC 3.2.1.7), xylanases (EC 3.2.1.8) andchitinases (EC 3.2.1.14), respectively, produced by members ofthis genus. The details of carbohydrases produced byNocardiopsis species are summarized in Table 1.

Fig. 1 Summary of the extremeenvironmental conditions whereNocardiopsis species areprevalent

Appl Microbiol Biotechnol

Starch is an abundantly available natural polymer of D-glucose monomers linked via α(1→4)-glycosidic bonds. Avariety of amylases act on the glucosidic linkages present inthe biopolymer (Souza and Magalhães 2010). Nocardiopsisspecies inherently produce some α-amylases. The most im-portant feature regarding these enzymes is their occurrence inspecies isolated from extreme environments with particularreference to temperature. As discussed in the “Introduction”section, some species of this genus have been isolated fromcold regions, and they have been explored for the productionof cold-active amylases. During an investigation on the isola-tion of psychrotrophic bacteria with cold-adaptive amylolytic,lipolytic or proteolytic activities, bacterial cultures were iso-lated from deep-sea sediment of Prydz Bay, Antarctica (Zhangand Zeng 2007). The amylase-producing strains belonged togenus Pseudomonas, Rhodococcus and Nocardiopsis.Nocardiopsis strain 7326 obtained from this region yielded acold-adapted α-amylase optimally active at 35 °C under alka-line conditions (Zhang and Zeng 2008). The enzyme wasinactivated at temperatures above 45 °C. This enzyme degrad-ed starch to glucose, maltose and maltotriose. Other featuresof this enzyme are detailed in Table 1. Another cold-adapted

α-amylase was obtained from Nocardiopsis aegyptia initiallyisolated from marine sediment in Egypt (Abou-Elela et al.2009). The α-amylase was optimally produced at a relativelylower temperature of 25 °C under acidic conditions. Fromthese reports, it is evident that Nocardiopsis species livingunder cold conditions have evolved to produce cold-adaptedenzymes that enable their survival under such adverse condi-tions. It must be noted that such cold-active or cold-adaptiveenzymes are not only significant with respect to fundamentalscientific studies but are also relevant in industrial settings(Cavicchioli et al. 2011; Feller 2013). Like other cold-adaptedstarch-hydrolyzing enzymes, α-amylases obtained fromNocardiopsis species could find applications in bread making,textiles, brewing and detergents.

In contrast to the aforementioned cold-adapted α-amy-lases, a thermotolerant variant of the enzyme has been obtain-ed from an endophytic species of Nocardiopsis from Brazil(Stamford et al. 2001). The thermostability of the enzyme wasevident from the fact that it was completely unaltered at 70 °Cand retained 50 % of the activity when incubated at 90 °C for10 min. This thermostable enzyme could be put to use forpreparing high-fructose syrups. Such syrups are extensively

Table 1 Summary of the carbohydrases produced by Nocardiopsis species

Carbohydrase Species/Location Enzyme pH;temperature optima

Other featuresand applications

Reference

Cold-adaptedα-amylase

Nocardiopsis strain7326, Antarctica

8.0; 35 °C Stable between pH 5 and 10; inactivatedabove 45 °C; stimulated by Ca2+, Mn2+,Mg2+, Cu2+ and Co2+; inhibited by Rb2+,Hg2+ and EDTA

Zhang and Zeng 2008

Cold-adaptedα-amylase

N. aegyptia, Egypt – Fermentation conditions optimized byPlackett-Burman statistical design;enhanced production after whole cellimmobilization on luffa pulp

Abou-Elela et al. 2009

Thermotolerantα-amylase

Nocardiopsisspecies, Brazil

5.0; 70 °C Industrial applications involving hightemperatures

Stamford et al. 2001

Thermostableα-amylase

Nocardiopsis sp. B2 9.0; 45 °C Maximum enzyme activity with 11 % (w/v)NaCl. Retained 75 and 69 % activityafter incubation for 1 h at 75 and 85 °C,respectively. Effective immobilization ingellan gum microspheres.

Chakraborty et al. 2014

Thermoalkalotolerantcellulase

Nocardiopsis sp.KNU, Korea

5.0; 40 °C Generation of fermentable rice strawhydrolysates for ethanol production

Saratale and Oh 2011

Alkalotolerant cellulase Nocardiopsis sp.SES28, Argentina

8.0; 40 °C Application in the detergent industry Walker et al. 2006

Thermoalkalotolerantβ-1,3-glucanase

Nocardiopsis sp. F96,Japan

9.0; 70 °C Hydrolyzed insoluble β-1,3-glucans;preferred β-1,3-1,4-glucans rather thanβ-1,3-glucans; cloning and expression;crystal structure; construction andcharacterization of chimeras

Masuda et al. 2003, 2006;Fibriansah et al. 2006,2007; Koizumi et al. 2007,2009

Alkali-tolerantthermostableinulinase

Nocardiopsis speciesDN-K15, China

8.0; 60 °C Exoinulinase (fructose as hydrolysisproduct)

Lu et al. 2014

Thermotolerantxylanases

N. dassonvillei subsp.alba OPC-18,Japan

X-I and X-II, 7.0;60 °C X-III,50 °C

Xylan bioconversion into xylosefor different products

Tsujibo et al. 1990a, 1991

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used in the beverage industry as sweeteners for making softdrinks (Van der Maarel et al. 2002). Another recent paperdescribes the isolation and immobilization of a thermostableα-amylase derived from a haloalkaliphilic marine isolate re-ferred to as Nocardiopsis sp. B2 (Chakraborty et al. 2014).The enzyme immobilized in gellan gum beads could be re-leased in a sustained manner. This property was highlighted tobe valuable in the pharmaceutical industries particularly forpreparing digestive formulations wherein continued release ofthe enzyme over a period of time is desirable.

Cellulose, the most abundant organic polymer on Earth, iscomposed of D-glucose units linked through β(1→4)-glyco-sidic bonds, and cellulases are hydrolytic enzymes acting onthis polymer (Lynd et al. 2002). In a recent report, Andersonet al. (2012) have identified putative cellulose-degrading en-zymes in the genomes of some actinobacteria. Among the 11organisms evaluated, eight including Nocardiopsisdassonvillei IMRU 509 produced cellulases. Cellulase activitywas observed when azurine cross-linked hydroxyethyl cellu-lose was used, and increased levels of the enzymes wereparticularly demonstrated in the presence of cellobiose.N. dassonvillei genomes showed sequences for six predictedcellulases. Apart from this report on the predicted cellulases inN. dassonvillei IMRU 509, another isolate, Nocardiopsis sp.(KNU) obtained from a Korean soil sample produced a varietyof thermoalkalotolerant carbohydrases including cellulases(Saratale and Oh 2011). This strain was strongly cellulolyticand utilized carboxymethyl cellulose (CMC), avicel, cellobi-ose, filter paper and rice straw effectively. The organism grewoptimally (at 37 °C and pH 6.5) under static conditions andproduced cellulases that were thermotolerant andalkalotolerant. The authors have also reported that rice strawcould be effectively converted into hydrolysates by usingthese enzymes. Such hydrolysates were further used as sub-strates by a strain of Saccharomyces cerevisiae for ethanolfermentation. Another isolate from Argentina, Nocardiopsissp. SES28 produced cellulases when grown in the presence ofCMC (Walker et al. 2006). The culture produced the enzymewhen it was grown at pH 8.0. The enzyme retained most of itsactivity even at pH 10.0, and therefore, its suitability forapplication in the detergent industry was proposed.Cellulose-degrading enzymes are important in laundry opera-tions, in fabric industries and in the production of biofuelsfrom lignocellulosic biomass (Kuhad et al. 2011). On accountof these varied applications, further studies on cellulases fromdifferent Nocardiopsis species are warranted.

β-Glucans (polysaccharides of D-glucose linked by β-glycosidic bonds) are a diverse group of polymers. They showa great diversity with respect to molecular mass, viscosity,solubility and three-dimensional configuration (Laroche andMichaud 2007). They are mainly encountered in the cell wallsof certain fungi and the bran of cereal grains (Manzi andPizzoferrato 2000; Kerckhoffs et al. 2003). β-1,3-

Glucanases are a class of enzymes that can degrade thesebiopolymers. Decomposition of β-1,3-glucan is an importantprocess because there are industrial requirements for β-1,3-glucan hydrolysates (Laroche and Michaud 2007). Theseenzymes are also important in biocontrol of phytopathogenicfungi (Masih and Paul 2002). β-1,3-Glucan is a constituent offungal cell walls, and its hydrolysis by β-1,3-glucanases canweaken or damage the fungal cell wall.

An alkaliphilic Nocardiopsis sp. (F96) isolated in Japanproduced three endo-β-1,3-glucanase isozymes with varyingmolecular masses referred to as BglF1, BglF2 and BglF3(Masuda et al. 2006). The three isozymes shared N-terminalamino acid sequences and were thus deduced to be products ofa single gene (bglF). This gene was cloned, and the expressedBglF protein showed highest amino acid sequence homologywith the GHF16 family of endo-β-1,3-glucanases (Masudaet al. 2003). The enzyme was thermoalkalotolerant (optimumpH and temperature: pH 9.0 and 70 °C, respectively). Thehydrolysis of different substrates such as laminarin,pachyman, curdlan, lichenan, CMC and avicel was evaluated.The enzyme efficiently hydrolyzed insoluble β-1,3-glucanssuch as pachyman and curdlan and the fairly soluble laminar-in. However, it displayed highest activity towards lichenan, aβ-1,3-1,4-glucan. These results indicated BglF to be a novelendo-β-1,3-glucanase. Studies involving mutational analysissuggested that Glu 123 and Glu 128 could be the catalyticresidues of BglF (Masuda et al. 2006). This enzyme wassuccessfully crystallized by the hanging-drop vapour diffusionmethod (Fibriansah et al. 2006). In a later report, the authorshave presented the first crystal structure of this endo-β-1,3-glucanase at 1.3-A° resolution and have compared it with theother homologous structures to analyze its substrate prefer-ence. Results of these studies confirmed that the enzyme had acatalytic centre comprising of Glu 123 as the putative nucle-ophile and Glu 128 as the acid-base catalyst. Trp 118 wasfound to be extremely important in substrate binding(Fibriansah et al. 2007).

The β-1,3-glucanase was shown to have a single catalyticdomain. In order to enhance the enzymatic properties of BglF,additional carbohydrate-binding domains were introduced,chimera proteins were created, and they were characterized(Koizumi et al. 2007, 2009). Four chimeras containing BglFalong with the following carbohydrate-binding modules (i) C-terminal additional domain (CAD) ofβ-1,3-glucanase H fromBacillus circulans IAM1165, (ii) N-terminal additional do-main (NAD) of β-1,3-glucanase H from B. circulansIAM1165, (iii) both CAD and NAD and (iv) chitin-bindingdomain (ChBD) of chitinase from alkaliphilic Bacillus sp.J813 were constructed. These chimeras respectively referredto as (i) BglF-CAD, (ii) NAD-BglF, (iii) NAD-BglF-CAD and(iv) BglF-ChBD were further characterized with regard totheir binding with insoluble β-1,3-glucans and their hydrolyt-ic behaviour towards these polymers. BglF-CAD chimeras

Appl Microbiol Biotechnol

displayed better binding abilities and hydrolytic activitiestowards these insoluble glucans. In particular, the hydrolyticactivities of this chimera under alkaline conditions (pH 9.0–10.0) and higher temperatures (50–70 °C) were better than thenative protein. The chimera BglF-ChBD showed enhancedbinding and hydrolysis of the substrate pachyman, and relativeactivities of this chimera around 50–70 °C were also higherthan those of original BglF.

Wild-type and recombinant strains over-expressing nativeand chimeric β-1,3-glucanases generally find applications inthe biocontrol of fungi, in the production of yeast extract andin the clarification of wines (Cheng et al. 2013). The β-1,3-glucanases derived from Nocardiopsis were unusual sincethey acted on insoluble glucans.

Inulin is a natural carbohydrate reserve mainly observed inthe roots and tubers of plants such as Jerusalem artichoke,chicory and dahlia (Kango and Jain 2011). This polymerconsists of linear chains of β-2,1-linked D-fructofuranosemolecules that are terminated with glucose residues througha sucrose-type linkage at the reducing end. Acid- or inulinase-hydrolyzed inulin is used for the production of fructosesyrups, inulo-oligosaccharides and bioethanol (Negro et al.2006; Singh et al. 2007). Since acidic hydrolysis of inulinoften results in the formation of undesirable colour and by-products, the use of inulinases has become popular (Singhet al. 2006). Recently, an exoinulinase (β-D-fructanfructohydrolase) has been reported from Nocardiopsis speciesDN-K15 isolated from marine sediments in China (Lu et al.2014). The enzyme was alkali tolerant and thermostable andwas active over a wide range of pH (5.0–11.0). It retainedmore than 81 % of the activity after incubation at 60 °C for1 h. Like other inulinases, this enzyme could also be used toobtain high fructose syrups and inulinosaccharides.

Xylans are polysaccharides that mainly contain β-D-xyloseunits linked via β(1→4)-glycosidic bonds (Bastawde 1992).They are ubiquitous in nature, and several microorganismsincluding Nocardiopsis species produce xylanases, the en-zymes that catalyze the breakdown of this biopolymer. In thisregard, N. dassonvillei OPC-18 is reported to produce threetypes of xylanases. These enzymes named as X-I, X-II and X-III were purified and characterized (Tsujibo et al. 1990a).Although these enzymes were optimally active at pH 7.0,enzyme X-III retained almost 63 % of its activity even at pH11.0. Other features associated with these enzymes are shownin Table 1. The authors later deduced and compared the aminoacid compositions and partial N-terminal sequences of theenzymes (Tsujibo et al. 1991).

The whole genomes of N. dassonvillei and Nocardiopsisalba are sequenced. The N. dassonvillei genome shows theexistence of four predicted coding sequences (CDS) forxylanases: Ndas_2218, Ndas_2447, Ndas_2710 andNdas_3986 (Sun et al. 2010). The N. alba genome showsthe presence of one xylanases coding sequence, B005_4612

(Qiao et al. 2012). It must be noted that xylanases are impor-tant in the breakdown of xylan into xylose which in turn canbe used for producing different types of biofuels (Taherzadehand Karimi 2008).

Chitin is an abundantly available natural polysaccharidethat mainly acts as the supporting material of crustaceans,insects and fungi (Rinaudo 2006). It consists of 2-acetamido-2-deoxy-β-D-glucose monomers linked viaβ(1→4) linkages, and a group of enzymes, the chitinases,hydrolyzes this polymer (Ravi Kumar 2000). During a studyon the presence of chitin degraders from different sites, avariety of bacteria including those belonging to the genusNocardiopsis were obtained (Nawani and Kapadnis 2003).During the same period, Tsujibo et al. (2003) isolated analkaliphilic Nocardiopsis prasina strain OPC-131 in Japanthat secreted three types of chitinases (ChiA, ChiB and ChiBDelta). The genes encoding the first two types of chitinases(ChiA and ChiB) were cloned and sequenced. While ChiAhad amolecular mass of around 35 kDa and a catalytic domainhomologous with family 18 chitinases, ChiB was a 32-kDaprotein that contained the type 3 chitin-binding domain(ChtBD type 3) and a catalytic domain. The 32-kDa proteinshowed considerable similarity with the Streptomyces family19 chitinases. ChiB Delta, the third enzyme, was establishedto be the truncated form of ChiB (it lacked the ChtBD type 3domain). ChiB was more efficient in hydrolyzing chitin anddemonstrated better antifungal activity than ChiB Delta. Thissuggested that the ChtBD type 3 domain of ChiB played asignificant function in the effective hydrolysis of chitin and itsantifungal activity.

During another study, Apichaisataienchote et al. (2005)introduced ChiB gene in Streptomyces fradiae (an antagonistof Fusarium moniliforme). While the wild-type strain pro-duced only two chitinases, the recombinant secreted threechitinases including ChiB. The supernatant of the recombi-nant strain effectively inhibited hyphal growth ofF. moniliforme. The chitinase F1 (ChiF1) has been ob-tained from another alkaliphilic Nocardiopsis sp. (strainF96) from Japan, and its crystal structure has beenelucidated (Matsui et al. 2004). The enzyme belongsto family 18 chitinases.

The genome of N. dassonvillei shows the presence of threepredicted coding sequences for chitinases, namely,Ndas_0360, Ndas_0763 and Ndas_1329 (Sun et al. 2010).Moreover, B005_3231, B005_3232 and B005_3655 are thesequences for chitinases in N. alba (Qiao et al. 2012). Chitin-degrading enzymes and chitinolytic microorganisms in recentyears have gained importance (Brzezinska et al. 2014). In thefuture, chitinases are projected to be safe and sustainablealternatives for controlling pathogenic fungi. These hydrolyticenzymes also mediate efficient degradation of chitin in nature.The importance of Nocardiopsis chitinases has been recentlydemonstrated by following a metaproteomic approach

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(Johnson-Rollings et al. 2014). The authors foundNocardiopsis-like chitinases to be the main enzymes respon-sible for chitinolytic activity in the soil that was beinginvestigated.

Polyhydroxybutyrate depolymerase

Poly[(R)-3-hydroxyalkanoic acids] or poly(HAs) or PHAs area class of storage compounds that are produced by manybacteria during unbalanced growth. Several types ofhydroxyalkanoic acids have been identified as constituentsof PHA (Steinbüchel and Valentin 1995). They are projectedas eco-friendly alternatives to petroleum-derived plastics(Suriyamongkol et al. 2007). These compounds are biode-gradable because some bacteria have the enzymatic abilitiesof breaking down these polymers.

In this regard, Ghanem et al. (2005) isolated a strain ofN. aegyptia from marine seashore sediments in Egypt. Thestrain used 3-hydroxybutyrate (PHB) or its copolymers with3-hydroxyvalerate (PHV) in different proportions P(3HB-co-10–20 % HV) as the sole source of carbon. This was possibledue to its ability to produce extracel lular PHBdepolymerases (EC 3.1.1.75). The organism was moreeffective in hydrolyzing copolymers than the homopol-ymer. A statistical experimental (Plackett-Burman) strat-egy was employed to standardize the conditions formaximum activity. The main factors that affected theprocess positively were the content of sodium gluconate,volume of medium/flask and age of inoculum. Theorganism degraded polymeric films as evidenced bythe numerous irregular erosion pits seen in scanningelectron micrographs.

The thermoplastic properties and biodegradability of PHAshave made them important from the industrial point of inter-est, and bacteriologically produced PHB and its co-polymersare commercially available over the past decade. This subse-quently highlights the requirement for microorganisms pro-ducing enzymes that degrade PHB and its co-polymers, inorder to facilitate their degradation in an environment-friendlyprocess. Nocardiopsis species producing PHB depolymerasesare, therefore, very important in this regard. Although theproduction of PHB depolymerase has been experimentallyvalidated in N. aegyptia, the genomes of N. dassonvilleiDSM 4311 and N. alba show the presence of predicted codingsequences (CDS) for such enzymes. In N. dassonvillei,Ndas_0048 and Ndas_2245 and, in N. alba, B005_1932 arethe predicted coding sequences for PHB esterases (Sun et al.2010; Qiao et al. 2012). There is a need to investigate suchspecies for depolymerase activities and optimize parametersfor their production or genetically manipulate them if inherentlevels are low.

Features of proteases produced by Nocardiopsis species

Several microorganisms produce extracellular proteases thatfacilitate breakdown of complex proteins to simpler peptides,thereby providing a more accessible source of nutrition (Turk2006). Different species of Nocardiopsis produce a variety ofproteases, somewith unique biochemical properties, structuralfeatures and a range of applications as detailed in Table 2.

Most of the Nocardiopsis species produce extracellularalkaline serine proteases (EC 3.4.21). For example, aJapanese group studied an alkalophilic strain ofN. dassonvillei subspecies prasina OPC-210 with respect tothe purification and characterization of two types of extracel-lular alkaline serine proteases, designated as NDP-I and NDP-II (Tsujibo et al. 1990b). Both the enzymes displayed anoptimum pH in the alkaline range, and optimum temperatureswere 70 and 60 °C, respectively, for NDP-I and NDP-II. NDP-I was stable between a pH range of 4.0 to 8.0, and NDP-II wasmore alkali stable (pH 6.0 to 12.0). On the basis of amino acidcompositions and N-terminal partial sequence similarity stud-ies, NDP-I was classified as a chymotrypsin-like serine pro-tease, and NDP-II was classified as an aqualysin-like serineprotease (Tsujibo et al. 1990c).

Nocardiopsis NCIM 5124 isolated from an oil-pollutedtropical marine environment near Mumbai, India (Dixit andPant 2000a) and recently identified as N. dassonvillei on thebasis of 16 s rDNA sequence similarities (unpublished results)also produced two types of alkaline serine endopeptidasesreferred to as proteases I and II (Dixit and Pant 2000b). Boththe alkaline proteases demonstrated collagenolytic and fibri-nolytic activities, and protease I could also degrade elastin.Proteases with such unusual substrate specificities are veryimportant in medical and other biotechnological applications.It must be noted that thromboses can lead to fatal conditionssuch as myocardial infarction. Fibrinolytic agents of clinicalsignificance are mostly plasminogen activators. However,such activators display certain undesired side effects, exhibitlow specificity for fibrin and are relatively expensive (Kimet al. 2006). Fibrinolytic enzymes are being regarded as im-portant alternatives in thrombolytic therapy (Phan et al. 2011).Collagenases have potential applications in the leather indus-try in enhancing dye penetration and also in the environment-friendly bioconversion of collagen wastes (Kanth et al. 2008;Duarte et al. 2014). The ability to degrade elastin, however, issuggestive of pathogenic properties, as actinomycetes withthis ability can cause mycetoma (Lacey and Goodfellow1975). Proteases I and II derived from NCIM 5124 wereclassified as alkaline serine endopeptidases, apparently be-longing to the group of chymotrypsin-like serine proteasescharacterized by broad S1 subsite specificity as discussed byPerona and Craik (1997). Both proteases required extendedsubstrate binding for efficient catalysis. Although the twoproteases were similar in their overall properties and

Appl Microbiol Biotechnol

secondary structure characteristics, the results of substratespecificity studies suggested that they could differ with respectto their active site geometry. More recently, structure-functionstudies on protease I (designated as NprotI) have revealed thatthe polyproline II (PPII) fold, an unusual structural element,was responsible for imparting kinetic stability to the protein(Rohamare et al. 2013). This is the first report on a non-structural protein of microbial origin displaying the PPII helix(the details regarding this are discussed later in this review).

There are twomore reports on proteases fromNocardiopsisspecies of Indian origin. An isolate obtained from a limestonequarry in Northeast India identified as N. prasina HA-4 pro-duced alkaline proteases (Ningthoujam et al. 2009). An alka-line protease from a salt-tolerant alkaliphilic N. alba strain(OK-5) of Indian origin has been reported (Gohel and Singh2012). In addition to the features listed in Table 2, the enzymedisplayed a shift in temperature optimum from 70 to 80 °C inthe presence of 4 M sodium chloride and 30 % sodiumglutamate. The presence of sodium glutamate, H2O2, β-mercaptoethanol and different surfactants enhanced the activ-ity. Both these reports (Ningthoujam et al. 2009; Gohel andS ingh 2012) have sugges t ed the use o f theseNocardiopsis-derived proteases as detergent additives.

Some Nocardiopsis species of Brazilian origin are alsoreported to produce proteases with distinctive features.Moreira et al. (2002) have described the production of analkaline serine protease with features listed in Table 2. Theenzyme derived from this species was marginally inhibited bythe surfactant sodium cholate, unaffected by Tween 80 andstimulated by saponin, sodium dodecyl sulphate (SDS) andTween 20. This serine protease was bleach-stable and retainedactivity in the presence of high concentrations (10 %v/v) ofH2O2. The protease also maintained 50 to 60 % of its activityin the presence of commercial detergents. The authors havesuggested the application of this Nocardiopsis-derived prote-ase as a detergent additive. An extracellular proteolytic en-zyme preparation from another Nocardiopsis species isolatedfrom a Northeastern Brazilian soil sample displayed milk-clotting properties (Cavalcanti et al. 2004). It must be notedthat microbial enzymes acting on milk proteins may provideattractive alternatives to the conventionally used enzymechymosin (Hashem 1999; Jacob et al. 2011). The milk-clotting enzyme from Nocardiopsis species was partially pu-rified, and it displayed optimal milk-clotting activity at pH 7.5and 55 °C. Further, the authors have optimized conditions formaximum production of the enzyme (Cavalcanti et al. 2005).

Table 2 Summary of the proteases produced by Nocardiopsis species

Enzyme produced Species/Origin Molecular mass(kDa); pH;temperatureoptima

Methods ofpurification

Other features andapplications

Reference

Alkaline serineproteases

N. dassonvilleisubspeciesprasina OPC-210, Japan

Protease I, 21; 10.0to 12.0; 70 °Cand protease II,36; 10.5; 60 °C

Acetone precipitation,DEAE-SephadexA-50, CM-SepharoseCL-6B, SephadexG-75, phenyl-Toyopearl650 M columnchromatography

Isoelectric points (pI):protease I 6.4 andprotease II 3.8

Tsujibo et al. 1990b, c

Protease I: chymotrypsin-like

Protease II aqualysin-like

Alkaline serineproteases

N. dassonvilleiNCIM 5124,India

Protease I, 21 Protease I: on CM-Sephadex chromatography

pI protease I (NprotI), 8.3 Dixit and Pant 2000a;Rohamare et al. 2013

Protease II 23;10.0–11.0; 60 °C

Protease II: DEAE-cellulose, SephadexG-50, phenyl Sepharose,hydroxyapatitechromatography

Protease II, 7.0 NprotIdisplayed uniquepolyproline II helix(PPII)

Alkaline proteases N. prasinaHA-4, India

Not reported; 7.0and 10.0; 55 °C

Not reported Suitable as detergentadditive

Ningthoujam et al. 2009

Alkaline serineprotease

N. alba OK-5,India

20 kDa; 10.0; 80 °C Hydrophobic interactionchromatography

Enhanced activity in thepresence of Na-glutamate,H2O2, BME and surfactants

Gohel and Singh 2012

Alkaline serineprotease

Nocardiopsisspecies, Brazil

Not reported; 10.5;50 °C

Not reported Suitable as detergent additive Moreira et al. 2002

Milk-clottingprotease

Nocardiopsisspecies, Brazil

Not reported; 7.5;55 °C

Ammonium sulphateprecipitation andDEAE-cellulosechromatography

Milk clotting proteases Cavalcanti et al. 2004

Keratinase andprotease

Nocardiopsisspecies SD5,India

30 and 60; 9.0;50 °C

Not reported Suitable in featherwaste management

Saha et al. 2013

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In particular, soybean flour favoured the induction, synthesisand secretion of the protease. In a later report, this group hasdeveloped liquid-liquid extraction methods involving re-versed micelle and aqueous two-phase systems for recoveringthe aforementioned milk-clotting protease (Porto et al. 2005).It must be understood that reversed micelles are typicallyaggregates of surfactants in organic solvents wherein thealiphatic chains are directed towards the exterior and the polarheads towards the interior. The resultant polar nuclei contain-ing an aqueous microphase can be used to extract and solubi-lize hydrophilic compounds including proteins that can beback-extracted during a later step (Lazarova and Tonova1999; Krishna et al. 2002).

Microbial keratinases are proteolytic enzymes degradingrecalcitrant and insoluble structural proteins present infeathers, hair and nails. Some keratinases can also degradeprion proteins (Gupta et al. 2013a, b). They are generally usedin the dehairing of hides, keratin waste management, pharma-ceuticals and prion decontamination. There are a few reportson some Nocardiopsis species producing keratinolytic en-zymes. An alkaliphilic N. alba strain (TOA-1) isolated froma tile joint in Japan produced a variety of alkaline hydrolyticenzymes (Mitsuiki et al. 2002). Among these, NAPase(N. alba protease A), an acid-resistant, kinetically stable pro-tease, was studied in detail. The novel feature of this enzymewas that it was stable under acidic conditions. The role ofkeratinase such as KerA derived from Bacillus licheniformisPWD-1 in the degradation of prion proteins was first demon-strated by Langveld et al. (2003). NAPases could also effec-tively degrade the scrapie prion PrPSc without any prior chem-ical or physical treatment (Mitsuiki et al. 2006). NAPase whenincubated with the prion protein at 60 °C and at pH >10.0brought about its degradation within 3 min. It must be notedthat prion proteins are released into the environment throughbody fluids, disposal of cadavers, contaminated effluents fromslaughterhouses, hospitals and research facilities, and theypose potential health hazards (Bartelt-Hunt and Bartz 2013).The chemical and physical methods available for their disin-fection and sterilization are often harsh, energy-intensive anddo not ensure complete loss of infectivity (Rutala and Weber2010). In this regard, the use of enzymes such as NAPase mayprovide an eco-friendly alternative. The structural basis un-derlying the acid stability of this enzyme was determined byKelch et al. (2007) and is discussed later.

In a more recent report, a thermoalkaliphilic strain ofNocardiopsis sp. (SD5) was isolated from feather wastes inIndia (Saha et al. 2013). The crude preparations contained twoproteases that exhibited keratinolytic and proteolytic activitiesas detailed in Table 2. The role of this strain in controllingfeather waste pollution was suggested. It is well documentedthat feathers are a major contributor towards poultry wastes,and these can be treated with keratinolytic enzymes to yieldkeratin hydrolysates that are rich in nitrogen content and

hydrophobic amino acids (Gupta and Ramnani 2006;Brandelli 2008; Brandelli et al. 2010). Keratin hydrolysatesare useful products that can be used in diverse fields (Guptaet al. 2013a, b).

The potential for developing commercially viable productsfrom Nocardiopsis species (particularly with respect to prote-ases) is evident from some patents that have been granted todifferent investigators. There are patents on the use of theseenzymes in making yeast extracts and casein hydrolysates(Kalum 2008; Lynglev and Nielsen 2009). There is also onepermitting their recombinant production and use in animalfeed and detergents (Lassen et al. 2010).

Structural features of stable enzymes producedby Nocardiopsis species

Some of the enzymes produced by Nocardiopsis speciesdisplay unusual stabilities under different conditions, and thestructural features responsible for such behaviour have beendetailed. Most of these studies are related to proteases. In twocases, proteases derived from Nocardiopsis species are kinet-ically stable proteins (Kelch et al. 2007; Rohamare et al.2013). It must be noted that in such proteins, stability isdetermined only on the basis of kinetic barriers and not bythermodynamic equilibria. Such proteins have high freeenergy kinetic barriers separating the folded and unfoldedstates, and they tend to exist in the folded state even underharsh conditions.

Kelch et al. (2007) have described the unfolding character-istics of the acid-resistant, kinetically stable protease A(NAPase) derived from N. alba as detailed in this paragraph.The authors have mademost comparisons of this enzyme witha neutrophilic homolog (α-lytic protease, alphaLP). Althoughboth the enzymes (NAPase and alphaLP) had a similar num-ber of acid-titratable residues, on the basis of kinetic studies, itwas concluded that the height of the unfolding free energybarrier for NAPase was less sensitive to acid than that of itsneutrophilic counterpart (alphaLP). This attribute preservedthe activity of NAPase under acidic conditions. Further as-sessment of the structural details of the two enzymes identifiedthe relocation of multiple salt bridges (inherently present inthe domain interface of alphaLP) to outer regions in NAPase.An acid-stable form of alphaLP (wherein a single interdomainsalt bridge was replaced with a corresponding NAPase-likeintradomain salt bridge) displayed an exceptional (>15-fold)increase in acid resistance. This study based on NAPase, as anexample, has highlighted the significance of kinetic stabilityas an evolutionary means of overcoming extreme conditions.

More recently, NprotI, the protease derived fromN. dassonvillei NCIM 5124, was shown to display unusualfeatures (Rohamare et al. 2013). Structural and functionaltransitions of NprotI in the presence of chaotropic agents,

Appl Microbiol Biotechnol

alcohols and proteases and at high temperatures were studiedby employing a variety of biophysical and assay-based tech-niques. Circular dichroism spectra of the native protein, struc-tural variations in presence of guanidine hydrochloride and adistinct isodichroic point under denaturing conditions indicat-ed the presence of a distinctive polyproline II helix (PPII) inthe protease. It must be noted that the polyproline II structureis encountered in proteins when sequential proline residues arepresent. This helix is mainly present in collagen and somesegments or subunits of other proteins (RNA polymerase II,wheat glutenin, titin and dehydrins). As discussed above in thecase of NAPase, kinetically stable proteins are not easilyunfolded and resist denaturation by SDS and proteolytic di-gestion. The authors have discussed that the unusual stabilityof NprotI towards high concentrations of denaturing agents,organic solvents and proteolytic enzymes makes this enzymean interesting candidate for structural investigations. The sta-bility was attributed to the presence of PPII helix observed forthe first time as a global conformation in a non-structuralprotein of microbial origin. The PPII fold of the enzyme wasfound to be more stable towards chemical denaturants andproteolytic enzymes than to physical denaturants like heat,which could be assigned to the fact that PPII helix does nothave any internal hydrogen bonds for stabilization. Heat couldas well be disrupting the other stabilizing non-covalent inter-actions. The chemical reagents on the other hand might befulfilling the need of PPII helix for hydrogen bonding. Thisunusual stability of NProtI seems to be an example of adistinct attribute that nature has imparted on a Nocardiopsisspecies to enable its survival under harsh conditions.

The molecular basis underlying the stability of serine pro-tease derived from N. prasina has also been elucidated byanalyzing 121 multiple point mutation-containing mutant en-zyme clones (Farrell et al. 2012). Fast residual activity assays,a feature classification algorithm and structure-based energycalculation algorithms were used to understand the structuralfeatures. On the basis of multivariate regression analysis of the

data obtained from suchmutant clones, the significance of tworesidues (Asn 47 and Pro 124 situated in loop regions thatconfer stability to a highly homologous α-lytic protease) wasunderstood.

On the basis of the literature assessment on the subject andthe detailed discussion in the previous sections, it is obviousthat different species of Nocardiopsis are a potential source ofdiverse and novel enzymes. Among the few Nocardiopsiscultures that have been identified to the species level, it isevident that some produce more than one kind of novelenzymes as detai led in Table 3. Two strains ofN. dassonvillei produce extracellular alkaline serine proteases,and one produces a thermotolerant xylanase.N. alba producestwo types of proteases: an acid-stable keratinolytic enzymeand an alkaline protease. N. prasina strains are endowed withenzymes such as alkaline proteases and three types ofchitinases. N. aegyptia strains produce two diverse novelenzymes, a cold-adapted α-amylase and PHB depolymerase.It is also seen that Nocardiopsis species producing a range ofnovel enzymes have been obtained from specific geographicallocations. Particularly, such cultures have been isolated fromSouth America (Argentina, Brazil), Asia (China, India, Japanand Korea), Africa (Egypt) and Antarctica. On the basis oftheir location of isolation, there is a difference in the featuresof the enzymes produced. For example, the isolate fromAntarctica produces a cold-adaptedα-amylase and those fromtropical regions produce thermostable enzymes. The mostprevalent feature shown by these enzymes is their alkalitolerance and thermostability. Cellulases, glucanases,chitinases and proteases are enzymes that show these features(Tables 1 and 2). The wide range of enzymes allows theorganisms to survive under extreme environmental condi-tions. In some cases, the structural details responsible for theunusual stability of these enzymes have been elucidated.

In conclusion, Nocardiopsis species with unique physio-logical properties have been isolated from diverse locales, andseveral research workers address issues related to enzymes

Table 3 Enzymatic abilities ofsome selected species ofNocardiopsis

Strain Enzymes Reference

N. dassonvillei OPC-210 Alkaline serine proteases Tsujibo et al. 1990b

N. dassonvillei NCIM 5124 Alkaline serine proteases Dixit and Pant 2000a, b;Rohamare et al. 2013

N. dassonvillei subsp. alba OPC-18 Thermotolerant xylanases Tsujibo et al. 1990a

N. alba Acid-stable protease Kelch et al. 2007

N. alba strain (OK-5) Alkaline protease Gohel and Singh 2012

N. prasina Proteases Farrell et al. 2012

N. prasina OPC-131 Chitinases Tsujibo et al. 2003

N. prasina HA-4 Proteases Ningthoujam et al. 2009

N. aegyptia PHB depolymerase Ghanem et al. 2005

N. aegyptia Cold-adapted α-amylase Abou-Elela et al. 2009

Appl Microbiol Biotechnol

associated with these isolates. The biochemical diversity ofthe genus is evident from the range of novel enzymes that itproduces. The array of novel enzymes produced by this mi-crobe may be responsible for its recurrent incidence in a rangeof habitats. The organism secretes carbohydrases such asamylases, cellulases and glucanases that act on glucose-containing polymers. Inulinases and xylanases are the othercarbohydrases obtained from this genus. Chitinases and PHBdepolymerase, in turn, can have a wide range of biotechno-logically relevant applications, and the variety of proteases itproduces is commendable. Certain processes involving themembers of this genus have the potential to be scaled-up,and patents on such methods have been granted. In recentyears, several novel species have been reported from a varietyof geographical locations, and investigations on the enzymaticcapabilities of these isolates would allow the range of en-zymes to be expanded in the years to come.

Acknowledgments All authors thank the University Grants Commis-sion for funding under UPE Phase II. TB thanks CSIR, India, for JuniorResearch Fellowship.

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