UNIVERSITI PUTRA MALAYSIA

PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE XYLANASE CLONED FROM Bacillus coagulans ST-6

YOW GEOK YONG

FSAS 1997 16

PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE XYLANASE CLONED FROM Bacillus coagulans ST-6

By

YOW GEOK YONG

Thesis submitted in Fulfilment of the Requirements for the Degree of Master of Science in the Faculty of Science and Environmental Studies,

Universiti Pertanian Malaysia.

March 1997

ACKNOWLEDGEMENTS

I would like to express my most sincere thanks and appreciation to my

Chairperson, Assoc. Prof. Dr. Abdullah Sipat, Department of Biochemistry and

Microbiology, Universiti Pertanian Malaysia, for his guidance, encouragement and

constructive suggestions throughout the course of study. I also wish to extend my

thanks to Assoc. Prof. Dr. Khatijah Yusoff and Assoc. Prof. Dr. Mohd. Arif Syed for

their support and valuable discussion in making this project a success.

I am also very grateful to all the members in the Biotechnology Fundamental

Laboratory room 202, friends in Rumen Microbiology Laboratory, UPM, and many

others for all their help, cooperation and making my time in UPM an enjoyable and

memorable one.

To my beloved parents, brothers and sister, thank you for being understanding

and supportive during my entire study in UPM.

11

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS................................... ............................................... . . 11 LI ST OF TABLES................................................ .................. . . . .......... . . . ... ...... ..... . . Vll LIST OF FI GURES............ ..... ...... ............................ . ........................... ............ .. . .. Vin LIST OF PLATES............................................... .... ....... .... ......... ........................... IX LIST OF ABBREVI ATI ONS.... ..................................... ....................................... x ABSTRACT.............. .................... ..................................................................... ... xu ABSTRAK... ..... ...... ......... .......... ........................................................ ................... xiv

CHAPTER

I INTRODUCTI ON........................................................... ........................... 1

I I LITERATURE REVIEW .................................. .......................................... 3 Xylan: Chemical Structure.............................................................. . ........... 3 Xylan in Plant Cell Wall............................................................................. 6 Occurrence and Role ofXylanase............. . ................................................. 6 Classification of Xylanase. ..... ..... ... .................... ............... .......................... 7 Multiplicity of Xylanase......... .......... ................................ ........................... 10 Applications ofXylanase.......... . .. ............................................................... 1 1 Biochemical Studies of Xylanase... ......... . . .. . . . . . . . . . . . . .. . . . . . . . . . .. . . ............. . ...... 13

Purification ofXylanase................................................................. 13 Characterization ofXylanase................ ............... ........................... 17

Molecular Cloning of Xylanase............. ...................... ..... ...... ..... ............... 27 Xylanase from Xylanolytic Plasmid pBNX ........................................... M... 30 Objective..................................................................................................... 32

III

Page

I I MATERIALS AND METH ODS.......... . ...... . . ...... . ..... . . ................. .......... . 33 Chemicals.. ... ................... .................. ............................... ........................ 3 3 Organisms and Culture Conditions..... . . . . . . ..... . . . ... ..... ... . .. . . . . . . . ..... . .. . ......... 34 Preparation of Working Stock Culture.. . . . . . . . . . . . . . .. . . .... . . . . ..... ........ . . . . .. . . ... .. 34

Isolation of Bacteria.... . . . . . . . . . . . ....... . . ...... .... . ...... .. . . . . ... .. . ..... ..... . ..... 34 Expression of Xylanase Activity.... .... . . . .. . ... ...... ... ... . . ....... . . . .... ..... 35 Stock Culture Preparation.. ..... .. .... . ... .... ... . .. ... . . .... . . . . . .... ... . ..... ...... 35

Localization of Xylanase in E. coli HB 1 0 1 (pBNX) . ... ..... . ....... . . .. . ..... .... , 36

Preparation of Bacteria Culture ..... .... . ......................................... 36 Recovery of Extracellular, Periplasmic and Cellular Enzymes.... 36

Measurement of Xylanase Activity........ .................. . ................... . . ........ ... 37 Determination of Protein Concentration.. . . ... . ......... . ... ...... ............ . . . ....... . . 38 Purification of Xylanase.... . . . . . . . . . .. . . ... . . . ......................... . . . . .... . . ................. 39

Ultrafiltration.............. ..................... . . . .. . ....................... ....... ........ 3 9 Gel Filtration: Sephadex G-50 ...... . . ..... ............ . . . . .......... . . . ........ '" 40

SDS-Polyacrylamide Gel Electrophoresis Analysis................................. 41 Preparation of Resolving Gel.. . ... ................. ,. ....... . ............. .......... 4 1 Preparation of Stacking GeL.. ... ................ . .................................. 42 Sample Preparation.. ........ . . .. ........................................... .............. 42 Running Conditions....... .. . .......... . . .... . . ... .............. ..................... .... 43 Protein Staining....... . .............. ............ .. . . .. . .. ............... ............... . . . 43

Zymogram Analysis of Xylanase.............. ... . . ........ ......... . . .............. ....... ... 43 Characterization of Xylanase.......... ............ ................... . . . . ............ . ... . . . .... 45

Molecular Weight Determination................................................. 45 I soelectric Point Determination................................. ................... 45 Determination Optimum Temperature............................ ............. 47 Determination of Temperature Stability....................................... 47 Determination of Optimum pH.. . . . ................... .................. . . . .... ... . 47 Determination of pH Stability. ...... ... ....... ...... ... ... . . .......... .. .. . ......... 48 Determination of Kinetic Parameters .... . ...... ............................ . ... 48 Substrate Specificity................. . . . . ... ... ........ .... ...... .......... . ... . . . ........ 48 End Product Analysis.. ..... ....... .......... . . ....... ..... ............................ . . 49 Effect of Some Metal I ons, EDT A, Urea and SDS

on the Measurement of Glucose Using Somogyi-Nelson Assay and DNS Assay...... .. . . . . . .. .... .............. ....... . ........................ 50

Effect of Some Metal I ons, EDT A, Urea and SDS on the Xylanase Activity... . . ........... . . . ........ ...... . . . . . ........................ 5 1

iv

Page

I V RESULTS AND DISCUSSI ON...................................................... ......... 53 Expression of Xylanase Production......................... .................................. 53 Subcellular Localization of Xylanase in E. coli HB 1 0 1 (pBNX)............ . . 53

Purification of Xylanase ............. .................... '" ......... . . . . ........... '" ............. 57 Ultrafiltration. ....... ..... ......... ..... ........... ...... ... ...... ............................ 57 Gel Filtration: Sephadex G-50................. . .................................... 57 SDS-P AGE Analysis................................... .................................. 59 Zymogram Analysis........................................ . .......................... .... 59 Summary of Xylanase Purification............ . ................... . .. . . . ........ .. 63

Characterization of Xylanase..................... .......... ........................... . ......... . 65 Molecular Weight and I soelectric Point......................................... 65 Optimum Temperature for Xylanase Activity...................... .......... 68 Therrnostability of Xylanase........... ...... ............... .......................... 70 Optimum pH for Xylanase Activity............................................... 70 pH Stability of Xylanase...................... .............. ............................ 70 Kinetic Parameters............................................ . . ..... ..................... 74 Substrate Specificity..... ........ . . ........ ................. . . ............................ 76 End Product Analysis......... ... ........................................................ 78 Effect of Some Metal I ons, EDTA, Urea and SDS

on Xylanase Activity.............. ...................................................... 78

V CONCLUSI ON.......................................................................................... 87

REFERENCES...................................................................................................... 91

APPENDICES........... . ............. ......... . ........................... ......................................... 103

APPENDIX A: Preparation of 2X Yeast Trypton (2YT) Broth Containing Ampicillin (50!J.g/ml).. ................. ................. 103

APPENDI X B : Preparation of2YT Agar Containing Ampicillin (50 Ilglm1)............. ......................................... 104

APPENDIX C: i) Preparation of Remazol Brilliant Blue (RBB)-Xylan... 1 05 ii) Preparation of 2YT Agar Containing RBB-Xylan

and Ampicillin.... .............................. .......... ......... ......... 1 06 APPENDI X D: Preparation of McI lvaine Buffer (PH 7.2)......................... 1 07 APPENDIX E: Preparation of Reagents for Somogyi-Nelson Method...... 1 08 APPENDIX F: Preparation of 50 mM Phosphate Buffer (PH 7.2)............. 1 09 APPENDIX G: Preparation of Stock Reagent for SDS-P AGE.................. 1 1 0

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Page

APPENDIX H : Silver Staining Protocol (Pharrnacia Biotech)........... ........ 1 1 2 APPENDIX I : Preparation of Reagents for Coomassie Blue Staining...... 1 1 4 APPENDIX J: Broad Range I soelectric Focusing Calibration Kit,

pH 3- 10 (Pharrnacia Biotech).............................................. 115 APPENDIX K: Preparation of Buffers at Various pH ............................. . .. 1 1 6 APPENDIX L: Dinitrosalicylic Acid (DNS) Assay .................................. _ 117

VITA......................................................................................... .............................. 1 1 8

LIST OF PUBLI CATI ONS................................................................... ................. 1 19

VI

LIST OF TABLES

Table Page

Single Step Purification ofXylanase Using Various Types of Chromatography........................................................................................... 18

2 Characteristics ofXylanases from Thermophilic Microorganisms . ...... .. ..... 19

3 Characteristics ofXylanases from Mesophilic Microorganisms ..... w o ........... 21

4 Molecular Cloning ofXylanase Genes from Bacillus .................................. 28

5 Subcellular Localization ofXylanase in E. coli (pBNX) .......... ................... 55

6 Purification ofXylanase from E. coli (pBNX) ............... ................ . ............. 64

7 Substrate Specificity of Purified Xylanase .......................................... ......... 77

8 Student's t Test of the Difference between the Mean Absorbance in the Presence and Absence of Some Metal Ions, EDT A, Urea and SDS (1 mM) on the Measurement of Glucose................................. ............ 82

9 Student's t Test of the Difference between the Mean Absorbance in the Presence and Absence of Some Metal Ions, EDTA, Urea and SDS (1 mM) on the Measurement of Enzyme Activity ............................... 85

10 Properties ofXylanase Purified from E. coli (pBNX) ................................. 88

vii

LIST OF FIGURES

Figure Page

1 General Structure ofXylan......................................................................... 4

2 Restriction Enzyme Mapping of Recombinant Plasmid pBNX.. ............... 31

3 Sephadex G-50 Chromatography ofXylanase............................................ 58

4 Determination of Molecular Weight........................................................... 66

5 Effect of Temperature on Xylanase Activity.............................................. 69

6 Thermostability ofXylanase....................................................................... 71

7 Effect of pH on Xylanase Activity.............................................................. 72

8 pH Stability ofXylanase............................................................................. 73

9 Lineweaver-Burk Plot of Xylanase............................................................. 75

10 Effect of Some Metal Ions, EDT A, Urea and SDS (1 mM) on the

Measurement of Glucose. ....... ........ ..... ..... ............ ................ ...................... 81

11 Effect of Some Metal Ions, EDT A, Urea and SDS (1 mM) on the Activity ofXylanase................................................................................... 84

viii

LIST OF PLATES

Plate Page

1 Expression of Xylanase Activity of E. coli (pBNX) on 0.2%

RBB-Xylan Agar Plate.............................................................................. 54

2 SDS-PAGE Analysis................................................................................. 60

3 A: Coomassie Blue Staining of the Agarose Gel After Electrophoresis.... 62 B: Zymograrn of the Xylanase on Xylan-agar............................................ 62

4 A: Determination of pl. ............................................................................. , 67 B: Zymograrn of the Xylanase on Xylan-agar............................................ 67

5 TLC Chromatogram of the Hydrolysed Products of Oat-Spelt Xylan....... 79

ix

LIST OF ABBREVIATIONS

The following abbreviations were used in the text:

CFU colony form unit

CM carboxymethyl

CMC carboxymethylcellulose

°C degrees centrigrade

Da dalton

DEAE diethylaminoethyl

DNA deoxyribonucleic acid

DNS dinitrosalicylic acid

EDTA ethylenediamine tetra acetic acid

hr hour

kb kilo basepair

kDa kilodalton

Km Michaelis Menten constant

L litre

rnA milliampere

M molar

mg milligram

mm minute

x

ml millilitre

mM millimolar

MW molecular weight

PAGE polyacrylamide gel electrophoresis

pI isoelectric point

RBB remazol brilliant blue

Rf relative mobility

rpm revolutions per minute

SDS sodium dodecyl sulfate

TCA trichloro acetic acid

TEMED N ,N ,N' ,N' -tetramethy l-ethy lenediamine

TLC thin layer chromatography

Ilg mIcrogram

III microlitre

Ilmol micromole

V volt

v/v volume per volume

Vmax maximal velocity of a enzyme reaction

w/v weight per volume

YT yeast trypton

Xl

Abstact of the thesis submitted to the Senate ofUniversiti Pertanian Malaysia in fulfilment of the requirements for the degree of Master of Science.

PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE XYLANASE CLONED FROM Bacillus coagulans ST-6

By

YOW GEOK YONG

March 1997

Chairman : Assoc. Prof. Abdullah Sipat, Ph.D

Faculty: Science and Environmental Studies

The recombinant plasmid pBNX contains a xylanase gene cloned from a

thermophilic Bacillus coagulans ST -6. It was found to produce a high level of

intracellular xylanase activity in Escherichia coli HB 101. This xylanase enzyme was

purified to homogeneity via a single step chromatography using a Sephadex G-50

column. SDS-PAGE analysis showed a single protein band having a molecular mass

of 20 kDa. Zymogram analysis using agarose gel electrophoresis showed a single

activity band for both crude and purified enzyme. Isoelectric point of the purified

xylanase was pH 9.3.

The optimum temperature and pH for xylanase activity was 55°C and pH 7.2

respectively. The enzyme was found to be stable and retained its activity after 30 min

incubation at 60°C. The enzyme showed a broad range of pH stability, in that it

retained its activity after a 1 hr incubation at pH 5 to pH 10.

xu

The apparent Km and Vmax of the enzyme was 2.18 mg/ml and 147.6 ).lmol

xylose/min/mg protein respectively using oat spelt xylan as the suostrate. The

purified enzyme was found to have very low CMCase activity after prolonged

incubation of 5 hr to 24 hr (less than 5% that of xylanase activity after 24 hr

incubation). No activity towards Avicel and filter paper was observed. No xylose,

xylobiose or arabinose was found in the end product analysis using TLC indicating

that the xylanase was a nondebranching endoxylanase.

The effect of various metal ions on the measurement of reducing sugar by

Somogyi-Nelson and DNS methods was examined. At 1 mM, Ca2+, Mn2+ and Pb2+

increased colour intensity of the standard glucose measured by DNS method while

Mg2+, Zn2+, Hg2+ and EDTA decreased the colour formation. For the Somogyi­

Nelson assay, only Mn2+ and Hg2+ decreased colour intensity of standard glucose

measured while other compounds showed no effect. A subsequent study on the effect

of metal ions on xylanase activity, taking into consideration of these results, showed

that only Hg2+ was inhibitory while other metal ions (Li+, K+, Na+, Ca2+, Mg2+, Co2+,

Mn2+, Cu2+, Zn2+, Pb2+ and Fe2), EDTA, urea and SDS had no effect.

Xlll

Abstrak tesis yang dikemukakan kepada Senat Universiti Pertanian Malaysia sebagai memenuhi syarat untuk mendapatkan ljazah Master Sains.

PENULENAN DAN PENCIRIAN ENZIM XILANASE TERMOSTABIL YANG DIKLON DARIPADA Bacillus coagulans ST-6

Oleh

YOW GEOK YONG

March 1997

Pengerusi : Prof. Madya Abdullah Sipat, Ph.D

Fakulti : Sains dan Pengajian Alam Sekitar

Escherichia coli HB101 (pBNX) , yang mengandungi plasmid rekombinan

pBNX xilanolitik yang membawa gen xilanase daripada Bacillus coagulans ST-6

tennofilik, didapati menghasilkan aktiviti xilanase intrasel yang tinggi . Enzim

xilanase in telah ditulenkan sehingga homogen melalui kromatografi menggunakan

turus Sephadex G-50. Analisis SDS-P AGE menunjukkan satu jalur protein yang

bersaiz 20 kDa. Analisis zimogram menggunakan elektroforesis agarose gel

menunjukkan satu jalur aktiviti untuk enzim kasar dan juga enzim tulen. Titik

isoelektrik untuk enzim tulen ialah pH 9.3.

Suhu dan pH optima untuk aktiviti xilanase adalah 55°C dan pH 7.2. Enzim

ini dapat mengekalkan aktivitinya selepas dieram selama 30 min pada suhu 60°C.

Enzim tulen ini juga didapati stabil pada julat antara pH 5 hingga 10.

xiv

Anggaran bagi nilai Km dan Vmax bagi enzim tulen ialah 2.l8 mg/ml dan 147.6

�mol xilose/minlmg protein dengan menggunakan xilan oat spelt sebagai substrat.

Enzim tulen didapati mempunyai aktiviti CMCase yang rendah selepas tempoh

pengeraman dipanjangkan daripada 5 jam hingga 24 jam (kurang daripada 5%

daripada aktiviti xilanase selepas 24 jam). Tiada aktiviti dikesan terhadap A vicel and

kertas turas. Tiada xilose, xilobiose dan arabinose dapat dikesan dalam produk

terakhir hidrolisis xilan dengan menggunakan TLC, menunjukkan bahawa enzim tulen

adalah j enis endo-xilanase.

Kesan pelbagai jenis ion logam ke atas penentuan gula penurun dengan kaedah

Somogyi-Nelson dan DNS telah dikaji. Pada kepekatan 1 mM, Ca2+, Mn2+ dan Pb2+

telah meningkatkan pembentukan warna oleh glukosa piawai dengan menggunakan

kaedah DNS, manakalaMg2+, Zn2+, Hg2+ dan EDTA telah merendahkan pembentukan

warna tersebut. Untuk kaedah Somogyi-Nelson pula, hanya Mn2+ dan Hg2+ didapati

merendahkan intensiti warna dalam pengukuran glukosa piawai manakala kompaun

yang lain tidak menunjukkan sebarang kesan. Seterusnya, satu kajian tentang kesan

beberapa ion logam ke atas aktiviti xilanase telah dijalankan, dengan mengambil kira

keputusan tersebut. Hasil diperolehi menunjukkan hanya Hg2+ saja merupakan

. aka!. I 1· (L·+ K+ N + C 2+ M 2+ C 2+ Mn2+ C 2+ perencat enzun man a IOn ogam am 1, , a , a , g , 0 , , U ,

Zn2+, Pb2+ and Fe2), EDTA, urea dan SDS tidak menunjukkan kesan.

xv

CHAPTER I

INTRODUCTION

Dwindling resources of food and fossil fuels have aroused considerable

interest in the utilization of renewable plant biomass (Han, 1 983). Cellulose and

hemicellulose are reported to account for more than 50% of plant biomass on earth.

The value of these renewable bioresource is estimated to contain total energy

equivalent to 640 billion tonnes of oil (Gilbert and Hazlewood, 1 993) . Xylan, being

the major component of plant hemicellulose found in the cell wall is estimated to

constitute more than 3 0% of the dry weight of plant polysaccharides (Joseleau et al . ,

1 992) .

Xylan is largely found as heterogeneous polysaccharides constituting of a p­

( 1 ,4) l inked D-xylopyranosyl backbone which is substituted with various side chains

(Joseleau et aI. , 1 992). B iodegradation of xylan to its monomer often involves

microbial xylanase. Bioconversion of xylan found in the lignocellulosic waste to

useful products is one of the potential applications of xylanase in the future

(Woodward, 1 984). Xylanase free of cellulase activity can be used as biobleaching

agent in the pulp and paper industry to help to replace the use of hazardous chemical

such as chlorine and chlorine dioxide (Wong and Saddler, 1 992). Other potential

1

2

applications ofxylanase enzyme were reported in food industry (Biely, 1985) and feed

production (Bedford and Classen, 1992).

Industrial application of xylanase has not been proven to be economically

feasible due to the lack of complete understanding of microbial degradation of xylan.

Hence, research in improving enzyme production by microorganisms and in

increasing the fermentation capabilities of the enzyme under extreme conditions used

in industrial process is essential (Wong et al., 1988).

This thesis is a report on the purification and characterization of a

thermostable xylanase produced by a recombinant Escherichia coli HB 1 0 1 harbouring

a xylanolytic recombinant plasmid pBNX. The xylanase gene in the recombinant E.

coli (pBNX) has been previously cloned from a thermophilic xylanolytic bacteria,

Bacillus coagulans strain ST -6 isolated from a local hot spring (Sharifah, 1990; Sipat

et al., 1995b, Norwati, 1996).

CHAPTER II

LITERATURE REVIEW

Xylan: Chemical Structure

Xylan is found as complex heteropolysaccharides in the plant cell wall

(Gilbert and Hazlewood, 1993). The structure ofxylan comprises of B-(1 ,4) l inked D­

xylopyranosyl polymer, substituted at various points by monosaccharides or short

oligo saccharides (Joseluau et al., 1 992). Unsubstitued l inear xylan (homoxylan)

compose exclusively by D-xylose polymer has also been isolated from esparto grass,

tobacco stalks and guar seed husk (Wong et al. , 1 988 ; Joseleau et al., 1 992).

Figure 1 shows the general structure of the xylan from various sources

(Joseleau et al., 1 992). Xylan accounts for 1 5 -30% of the dry weight of hardwood

(Angiospems) (Timell, 1 967; Sjostrom, 1 98 1 ; Wong et aI., 1 988) which is usually

acetylated at the OH group at C-2 and C-3 of the xylose residues (Fengel and

Wegener, 1 983). Acetylation occurs at about one per two xylose residues of the

hardwood xylan (Bastawde, 1 992). The backbone of the acetyled hardwood xylan is

additionally substituted with 4-0-methyl-a-D-glucupyranosyluronic acid. The 4-0-

methyl-D-glucuronic acid is often l inked with glycosidic bond at the C -2 of the D-

3

(OAc)

:O\\/�'V(o��\\/�O�

OH oR

R: a-D-GlcpA( 1-+2)Xyl...

4-0Me-a-D-GlcpA( 1-+ 2)Xyl...

a-L-Araf( 1-+3)Xyl...

a-L-Araf( 1-+2)Xy1...

�-D-Galp(1-+5)a-L-Araf( 1-+3)Xyl...

�-D-Xylp( 1-+ 2)a-L-Araf( 1-+3)Xyl...

a-L-Araf(I-+2, 1-+3 and 1.-.+2,3 Araf)J)(1-+3)Xyl... Feruloyl p.coumaroyl Lignin

Figure 1: General Structure of Xylan (after Joseleau et aI., 1992)

4

5

xylose residues, though C-3 linkages have also been observed (Whistler and Richards,

1970). Most hardwood xylan has approximately one acidic side chain per ten D­

xylose residues (Whistler and Richards, 1970). L-rhamnose and galacturonic acid are

also found to linked to the reducing end of hardwood xylan (Sjostrom, 1981; Fengel

and Wegener, 1983).

Xylan is less abundant in softwood (Gymnosperms) and accounts about 7-

12% of total dry weight of softwood (Wong et al., 1988). Softwood xylan differs

from hardwood xylan due to the lack of the acetyl group and the presence of L­

arabinofuranose units at an average of 1.3 residues per ten xylose units, linked by a­

(1�3) glycosidic bond (Sjostrom, 1981; Fengel and Wegener, 1983). The softwood

xylan is also partially substitued with 4-0-methyl-D-glucuronic acid groups at C-2, on

an average of two residues per ten xylose units (Sjostrom, 1981).

Xylan from other plants has also been investigated. There are no marked

structural differences between the D-xylan from cereals, grasses and wood (Whistler

and Richards, 1970). Besides the homoxy1an isolated from esparto grass, other

grasses like bamboo, barley and Guinea grass are found to be heteroxylan containing

arabino-4-0-methy1-glucurono-xylan substitution (Fengel and Wegener, 1983). The

cereal D-xylan is also found to have L-arabinose and D-glucuronic acid residues

attached to the main chain (Whistler and Richards, 1970). Xylan present in seaweed

is different from the terrestrial plants, for example the marine algae are found to

contain �-( 1,3 )-linked D-xylosyl backbone (Joseleau et aI., 1992).

6

Xylan in Plant Cell Wall

Ultrastructural localization studies of xylan in the plant wall showed that it

interconnects with other structural cell wall components via covalent linkages and

also non-covalent secondary forces forces (Wong et aI. , 1988 ; Joseleau et aI., 1992).

Lignin and some phenolic acids appear to covalently link to xylan via arabinosyl and

glucuronosyl residues (Wong et aI., 1988). Covalent linkages between xylan and

other xylan chains, galacturonan or pectin subtances have also been suggested. Xylan

and cellulose microfibrils are believed to interact via hydrogen bonding (Joseleau et

aI., 1992).

Both these covalent and noncovalent forces have contributed to the strength of

lignocellulose having structural functions (Wong et aI., 1988 ; Joseleau et al., 1992).

Xylan is also present in the primary wall of growing cells or seed and bulbs and

functions as food reserve (Joseleau et aI., 1992).

Occurrence and Role of Xylanase

Xylanase plays an important role in plant matter degradation by

microorganisms to provide a source of metabolizable energy. �-(1 �4)-D-xylanase is

reported to be produced by bacteria from marine and terrestrial environment, fungi,

rumen bacteria and protozoa, ruminant caecal bacteria, insects, snails, crustaceans,

7

marine algae, and germinating seeds of terrestrial plants. A P-(l -+3)-D-xylanase is

also found to be produced by some bacteria and algae from marine environment.

Some terrestrial fungi and bacteria are reported to produce both P-( 1 -+ 3) and P­

(l-+4)-D-xylanase. However, xylanase has not been found to be produced by

vetebrate animal tissue (Dekker and Richards, 1 976).

Ruminant depend on xylanase produced by rumen microbial flora and fauna

for the degradation of hemicellulose in its diet intake (Woodward, 1 984). Xylanase

has also been implicated in plant disease caused by bacterial or fungal plant pathogens

in which it assists in the infection process by causing disruption of cell wall barrier

(Woodward, 1984; Coughlan, 1992).

Classification of Xylanase

Xylanase enzymes can be classified according to their mode of action on the

xylan substrate. According to Reilly (198 1 ) and Bastawde (1 992), xylanase can be

classified as:

1 . Endo-p-(1 -+4)-D-xylanase [P-(1 -+4)-D-Xylan Xylano Hydrolase, EC 3.2.1.8]

The endo-xylanase attacks the glycosidic bonds within the xylan backbone to

produce xylooligosaccharides of various chain lengths. Cleavage is not random but is

determined by the length of the substrate, its degree of branching and presence of

subtituents. Endo-xylanase can be further divided into 4 types:

8

a) Non-arabinose Liberating Endoxylanase (Xylobiose and Xylose Producers).

This enzyme cannot cleave L-arabinosyl initiated branch points at �-(l �4)

linkages and produces mainly xylobiose and xylose as the end products. These

enzymes can break down xylooligosaccharides of size as small as xylotriose but

they are more active on larger substrates.

b) Non-arabinose Liberating Endoxylanase (Xylooligosaccharide Producers).

This enzyme cannot cleave branch points at a-(1�2) and a-(1�3) and produces

xylooligosaccharides larger than xylobiose as the major end products. It is

generally inactive on xylotriose and xylobiose.

c) Arabinose Liberating Endoxylanase (Xylobiose and Xylose Producers).

This enzyme cleaves xylan chain at the branch points and produce mainly

xylobiose, xylose and arabinose as the end products.

d) Arabinose Liberating Endoxylanase (Xylooligosaccharides Producers).

This enzyme cleaves branch points and produce mainly xylooligosaccharides of

intermediate sizes and arabinose.

The endo-xylanases are the enzymes for general xylan depolymerization and

have been extensively studied. A review on xylan and microbial xylanases by

Bastawde (1992) showed that endo-xylanases have a broad range of properties and

exhibit enzyme multiplicity.

9

2. Exo-�-(l44)-D-Xylanase [�-(l44)-D-Xylan Xylohydrolase]

An exo-xylanase removes single xylose units at a higher rate from the non­

reducing end of the xylan chain than short xylooligosaccharides and have no

transferase activity. Exo-xylanase is difficult to distinguish from �-xylosidase as both

of them are capable of degrading D-xylooligosaccharides. However, only exo­

xylanase can attack D-xylan.

Exo-xylanase has remained largely unstudied (Bastawde, 1992). These

enzymes deserve more emphasis in industrial use because of their advantageous

properties, such as; the absence of transferase activity, low inhibition by xylose and

also the ability to attack larger xylooligosaccharides (Reilly, 1981). Observation from

the products of hydrolysis has suggested the possible presence of exo-xylanase

component in an alkalophilic Bacillus sp. (Balakrishnan et ai., 1992) and Aeromonas

caviae ME-l (Kubata et ai., 1994).

3. �-xylosidase [Ee 3.2.1.37]

l3-xylosidase breaks down short xylooligosaccharides to xylose from the non­

reducing end, and have substantial transferase activity which decrease the yield of

xylose. These enzyme cannot degrade D-xylan substrate.