Chapter 6

Summary

SUMMARY

X ylan is a com plex and m ajor hetero polysaccharide constituent o f

hem icellulose, w hich is present in the form o f arabinoxylan in softw ood and grasses,

and as O-acetyl-4-O -m ethylglucurono-P-D-xylan in hardwood. T his heteropolym er

containing a backbone o f P-1,4 linked xylopyranosyl units is substituted w ith L-

arabinosyl, acetyl, feruloyl, p-coumaroyl and glucuronyl residues. Due to the

heterogeneity and com plex chem ical nature o f xylan, its hydrolysis into sim pler

constituents requires synergistic action o f w ide spectrum o f enzym es. M ost important o f

these enzym es is endoxylanase (EC 3.2.1.8) that cleaves P-1,4 linked xylose backbone,

w hile P-xylosidase (EC 3.2.1.37) hydrolyses xylo-oligom ers. In addition, a variety o f

debranching enzym es, i.e. a-arabinofuranosidase (EC 3.2.1.55), acetyl xylan esterase

(EC 3.1.1.72) and other hem icellulolytic enzym es, a-g lucuronidases (EC 3.2.1.139), a -

galactosidases (EC 3.2.1.22) and P-m annosidases (EC 3.2.1.25) are required for

effective utilization o f hem icellulosic fraction.

M any m icroorganism s, including bacteria, actinom ycetes as w ell a s fungi have

been investigated fo r their ability to produce endoxylanases that can degrade P-1,4-

xylan in random fashion, yielding a series o f linear and branched oligosaccharide

fragm ents. O f these therm ophilic/therm otolerant fungal strains are o f prim e importance

being source o f therm ostable enzym es, w hich are important for industrial applications

because o f possible econom ic benefits o f being able to carry out catalysis a t elevated

tem peratures. In recent years xylanases have received a great deal o f research attention

particularly because o f their biotechnological potential in various industrial processes

such as food, feed, biofuels and pulp & paper industries. Currently, the m ost prom ising

application o f xylanases is in the pre-bleaching o f pulps m ainly because o f a desire to

replace chlorine as a bleaching agent. Xylanases prom ote bleaching through the

hydrolysis o f relocated and re-precipitated xylan on the surface o f pulp fibre which

results in better chem ical penetration and consequently im proved lignin extractability.

This application requires that the xylanases should be active and stable at high

tem perature under alkaline conditions. Therefore search for novel m icrobial strains that

can produce h igh levels o f therm ostable alkaline active xylanases w ith an ability to

Summary

remove xylan from different kinds o f pulps is desirable. Owing to immense importance

o f alkaline active thermostable xylanases, this study was designed with the following

objectives:

• Isolation, screening and molecular characterization o f xylanase producing

thermophilic/thermotolerant fungi.

• Optimization o f xylanase production by novel fungal strain Malbranchea flava

using solid state fermentation (SSF) by one variable at a time and statistical

response surface methodology approaches.

• Study the regulation and expression o f xylanases and acetyl esterases by

Malbranchea flava.

• Purification and characterization o f xylanase isoforms and acetyl esterase from

Malbranchea flava.

• Evaluation o f the produced xylanases for bleaching o f the pulp.

6.1 Screening and molecular characterization o f therm ophilic/therm otolerant

fungi

Sixteen thermophilic/thermotolerant strains namely Acrophialophora nainiana

MTCC 6662, Aspergillus caespitosus MTCC 6326, Chaetomium thermophile MTCC

4891, Corynascus sepedonium MTCC 6490, Emericella nidulans var. lata MTCC

6327, Emericella nidulans var. nidulans MTCC 6339, Humicola fuscoatra MTCC 6329,

Humicola insolens MTCC 4617, Malbranchea flava 4889, Melanocarpus albomyces

MTCC 3922, Mucor indicus MTCC 6333, Myceliophthora sp. MTCC 6661,

Penicillium janthinellum MTCC 6564, Rhizomucor pusillus MTCC 4895, Thermoascus

aurantiacus MTCC 4890, and Thermomyces lanuginosus D2W3 isolated from

composting soils were included in the present study. These isolates were identified on

the basis o f morphological and molecular characteristics. The molecular

characterization was based on Amplified rDNA Restriction Analyses (ARDRA) o f the

18S rDNA as well as sequence analysis o f ITSI-5.8S-ITSII and D1/D2 hyper-variable

region of 26S rDNA. Thel8S rDNA amplified PCR product from different fungal

isolates ranged between 1.6-1.7 Kb in size was digested with Mbol, H infl and Rsal to

study the restriction pattern. The dendrogram constructed by combined cluster analysis

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Summary

o f restriction pattern obtained by M bo\, ///n fl and Rsal revealed th ree d istinct clades

w ith m em bers o f m ucorales, i.e., R. pusillus and M indicus form ing an ou t group. O ther

14 fungal strains w ere resolved in tw o distinct clades. The other clade w as characterized

w ith M. Jlava show ing an early divergence, while, M y celiophthora sp. and C.

sepedonium show ing h igh sim ilarity coefficient w ere clubbed together. The

phylogenetic trees constructed on the basis gene sequences o f ITSI-5.8S-ITSII region

and D1/D2 hyper-variable region o f 26S rDN A, show ed sim ilar topology. However,

ITS gene tree w as m ore robust as m ore nodes w ithin ITS tree received higher measure

o f support from bootstrapping. M oreover, the branch lengths show ed deeper nesting and

better resolution in ITS tree as com pared to D1/D2 hyper-variable region o f 26S rDNA.

The dendrogram based on ITS sequence analysis show ed that therm otolerant fungi

belonging to A spergilli and Penicillii and therm ophilic fungi w ere clustered separately.

Therm ophilic fungal isolates, i.e., T. lanuginosns, M. Jlava and M. albomyces,

producing alkaline active xylanases show ed m uch earlier phylogenetic divergence as

com pared to other isolates included in the study. All the therm ophilic and

therm otolerant strains w ere screened for xylanase production. T he results showed that

the strains included in the study produced m ultiple xylanase. Therm ophilic fungal

isolates T. lanuginosus, M. Jlava, and M yceliophthora sp., produced 9556, 3475 and

633.7 (U /g DW substrate) xylanase, respectively. The xylanases produced by these

three isolates w ere characterized to be alkaline active.

6.2 Biobleaching o f paper pulp

T he bleaching o f D ecker pulp w ith the xylanases produced by therm ophilic and

therm otolerant fungal strains w as carried out. The treatm ent o f pulp w ith xylanase from

M. Jlava resulted in m axim al release o f chromophores (237, 254 and 280 nm). The

released chrom ophores at these wavelengths w ere also high in the pulp treated with

xylanase from 71 lanuginosus, M. albomyces and A. caespitosus, though it was

appreciably less than that observed in M. Jlava. The increase in brightness o f the

resultant hand sheets obtained after treatm ent o f pulp w ith xylanases o f M. Jlava, 71

lanuginosus, M. albomyces and A. caespitosus w as 2.04, 2.07, 1.81 and 1.63% ISO

units, respectively.

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Summary

6.3 Optimization o f culture conditions for hemicellulase production by

M albranchea Jlava

A novel thermophilic fungal isolate Malbranchea flava which showed distinct

phylogenetic origin and found to be a good source o f xylanase. Based on its capability

to enhance brightness o f pulp by 2.04% ISO units, it was taken up for further

optimization o f culture conditions for improving the production o f hemicellulases. The

initial optimization by classical one variable at a time approach resulted in improved

levels o f hemicellulase production on solidified culture medium containing sorghum

straw (1 mm particle size) as a carbon source and casein (2%) as nitrogen source

inoculated with a spore suspension (1.2 *107/g substrate) at a moisture content o f 80%.

The optimal pH and temperature were observed to be 7.0 and 40°C, respectively. Under

these conditions the culture produced xylanase (14900 U/g DW substrate), P-xylosidase,

(9.9 U/g DW substrate), acetyl esterase (52 U/g DW substrate) and ri-L-

arabinofuranosidase (2.980 U/g DW substrate). Further optimization was carried out

using response surface methodology based on Box-Behnken design o f experiments to

study effect o f interactions between casein concentrations, inoculum age and inoculum

level as independent variables on the production o f hemicellulases. Regression analysis

showed that the R2 values computed for xylanase, P-xylosidase, acetyl esterase, and a-

L-arabinofuranosidase were 93.85, 93.79, 98 and 88.24%, respectively, showing their

fitness in predicting the effect o f casein concentration, inoculum age and inoculum level

on xylanase, p-xylosidase, acetyl esterase, and a-L-arabinofiiranosidase production

under SSF by M. flava. Furthermore significant F values for the model and insignificant

lack o f fit also showed the robustness o f the experimental design to explain the obtained

results. It was found that optimal enzyme activities, i.e., 16,078, 10.97, 68.91, 3.786

(U/g DW substrate) o f xylanase, p-xylosidase, acetyl esterase (AE) and a-L-

arabinofuranosidase, respectively, were produced on solidified culture medium. Using

combination o f one factor at a time and RSM optimization approaches, 2.26, 3.12,6.02

and 3.26 folds increase in the levels o f xylanase, P-xylosidase, acetyl esterase (AE) and

a-L-arabinofuranosidase, activities, respectively, when compared to those under

unoptimized conditions were achieved. The culture under optimal process conditions

produced, 8 functionally distinct xylanases, in addition 5 ,1 0 and 6, P-xylosidase, acetyl

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Summary

esterase (A E) and a-L-arabinofuranosidase isoforms, respectively, as revealed by

zymogram developed against native PAGE were observed.

6.4 Two dimensional electrophoretic profiling o f secretome of Malbranchea

Jlava

T he crude enzym e extract produced under optim ized and unoptim ized culture

conditions was resolved by 2DE. Seventy tw o distinct protein spots w ere detected in the

pi range o f 3.0-5.6. T he culture under optim al conditions produced spectrum o f proteins

that show ed d istinct differences in the num ber and intensity o f protein spots when

compared to the one grown under unoptim ized conditions. Expression o f few o f the

proteins w as up-regulated under the optim ized condition w hile few others were down-

regulated. T he partial secretom e characterization using peptide m ass fingerprinting

(LC/M S/M S) identified the protein spots as xylanases (GH11) and

cellobiodehydrogenase, cellobiohydrolase constituting the com ponents o f cell wall

degrading enzym es. In addition to these a m itochondrial and cytoplasm ic glycyl-tRNA

synthase protein and tw o hypothetical proteins w ere also identified.

6.5 Induction and regulation o f xylanase and acetyl esterase production in

Malbranchea JlavaT he induction o f xylanase and acetyl esterase w as studied in presence o f various

com plex carbohydrates (oatspelt xylan, rice straw , sorghum straw, w heat straw, wheat

bran, com cob, and bagasse), m onosaccharides, disaccharides, alcohols and glycerol as

carbon sources under shake flask cultures. The zym ogram s developed from the extracts

resolved on PA G E show ed differential expression o f m ultiple xylanases in presence o f

these carbon sources. T he results show ed that the presence o f oat spelt xylan resulted in

induction o f e igh t electrophoretically distinct xylanases ou t o f w hich three o f the

xylanase isoform s (Ic, Id and Ig) w ere constitutive as these w ere expressed even in

basal m edium devoid o f any sugar/carbohydrate. However, in presence o f com cob,

bagasse, rice straw , sorghum straw and w heat bran, five xylanase isoforms were

expressed. W hereas, in presence o f arabinose, cellobiose and fructose as carbon source

only three constitutive xylanase isoform s w ere expressed. On the o ther hand xylose as a

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Summary

carbon source supported the expression o f all five isoforms that were produced in

presence o f complex lignocellulosics (com cob, bagasse, rice straw, sorghum straw and

wheat bran). However, in presence o f glucose, glycerol, ethanol, methanol and propanol

four xylanase isoforms i.e., three constitutive and an additional inducible isoform (Ie)

was expressed. The addition o f monosaccharides, disaccharides, alcohols and glycerol

to xylan containing medium led to repression o f xylanase production. The repressive

effect o f arabinose addition however was most severe as it resulted in low xylanase

activity (12.75 units/mg protein) when compared to 85.65 (units/mg protein) observed

in presence o f xylan as sole carbon source. The zymogram showing the production

profile o f xylanase by M. flava on oat spelt xylan as carbon source revealed sequential

induction o f xylanases. The culture showed expression o f two constitutive isoforms (Ic,

Id) and an inducible isoform (Ih) after 24 h o f incubation. This was followed by

expression o f isoform (Ig) after 48 h. Next in the sequence was the expression o f

xylanase isoform (le) which was observed after 72 h o f incubation, whereas, after 96 h

o f incubation the culture expressed all eight xylanase isoforms.

The zymogram developed against extract resolved by PAGE showed that the culture

produced multiple esterases where five o f the isoforms (la, lb, Id, I f and lh) were expressed

constitutively in medium devoid o f any sugar/ complex carbohydrate. Maximal expression o f

seven esterase isoforms (la, lb, Ic, Id, Ie, I f and Ig) were produced in presence o f rice straw.

Glycerol as carbon source supported high expression o f esterase followed by fructose xylose

and glucose after 120 h o f incubation. The level o f esterase in presence o f arabinose, lactose,

cellobiose and alcohols was very low and was almost similar to that observed in basal

medium (control). However expression o f five acetyl esterase isoforms was observed in

presence o f all monosaccharides, disaccharides, alcohols and glycerol that were used in the

study. No repression in acetyl esterase activity was observed when fructose was added to the

medium. However addition o f other monosaccharides, disaccharides, alcohols or glycerol to

the xylan containing medium repressed the acetyl esterase production and the observed levels

of activity were almost similar to that observed in presence o f basal medium.

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Summary

6.6 Purification and characterization of xylanases from Malbranchea flava

Two m ajor alkaline active xylanases (MFX I and MFX II) with molecular

masses o f 25.2 and 30 kDa and p is o f 4.5 and 3.7, respectively were purified to

homogeneity using ion exchange and hydrophobic interaction chromatography. The

purified xylanases (M FX I and MFX II) showed specific activity o f 1800 and 961.53

pmol m in '1 mg protein*1 corresponding to 21.304 and 11.39 fold purification,

respectively. Liquid chromatography/mass spectrometry (LC-M S/M S) analysis o f

tryptic digests o f MFX I and MFX II revealed similarity to endoxylanases from T.

lanuginosus and Penicillium chrysogenum belonging to GH family 11 and GH family

10, respectively. The match score was found to be 114 in case o f MFX I, however it

was 58 in case o f MFX II.

6.6.1 Temperature, pH optima and stability

The xylanases (MFX I and MFX II) were optimally active at pH 9.0 and at

70°C, exhibited a half-life o f 4 h at 60°C, while at 70°C and at pH 9.0, MFX I and II

lost 73 and 53% o f their activity, respectively, after 30 min.

6.6.2 Effect o f metal ions and other compounds

Both the xylanases were found to be resistant to inhibition by metal ions. The

enzyme activity o f MFX I was promoted by EDTA and Ca2+, whereas, DTT, (3-

mercaptoethanol, Mg2+, SDS and Fe2+, Zn2+ and Mn2+ slightly reduced the activity. On

the other hand, M FX II was moderately inhibited by Zn2+ and Fe2+while its activity was

positively modulated by other metal ions and chemicals. Complete inhibition o f both

the purified xylanases was observed in presence o f N-Bromosuccinimide (NBS)

indicating the presence o f tryptophan residues at active site o f MFX I and MFX II.

6.6.3 Substrate specificity

The endo-activity o f xylanases was tested by reducing sugar assay on a number

o f complex substrates and it was found that MFX II was more substrate specific as

compared to MFX I. The purified MFX I showed maximal activity on rye arabinoxylan

(RAX) followed by wheat arabinoxylan (WAX) and oat spelt xylan (OSX), as

compared to that on birchwood xylan (BWX) as substrate. However, MFX II showed

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Summary

almost similar activity against different xylan types. Further MFX I showed low levels

of activity on lichenan, but MFX II was hardly active on this polysaccharide. MFX I

showed no activity against any of the pNP substrates tested, however, MFX II was

active on pNP cellobioside which is a characteristic property o f GH10 xylanases. The

purified xylanases MFX I and MFX II showed higher affinity towards larchwood xylan

(LWX) as evident from the comparatively lower Km values o f 1.2S and 3.7 mg/ml,

respectively. Kca/Km values suggested that the xylanases preferentially hydrolyzed

RAX. The results showed that 40% of the total purified MFX I adsorbed to avicel

(crystalline cellulose), however, MFX II did not show any adsorption to avicel

indicating putative absence o f carbohydrate binding module in MFX II.

6.6.4 Hydrolysis of xylan

The Thin Layer Chromatography (TLC) analysis showed that purified xylanases

(MFX I and MFX II) showed distinct mode o f action and hydrolysis product profiles

with BWX, OSX, LWX, as well as WAX and RAX. The resultant hydrolysis products

obtained with MFX I and II differed from each other. As the xylan degradation

occurred, a large amount o f intermediate products (X2, X3, X4 and X5) was obtained,

with a small amount of xylose (Xi). The hydrolysis o f BWX (which is primarily a

methylglucouronoxylan) with MFX I resulted in xylobiose (X2) as the major product

followed by xylotriose (X3) as the other main product. In addition, positional isomer

iso-Xs putatively identified aldopentauronic acid was detected which is a characteristic

property xylanase (GH11). The hydrolysis of BWX with MFX II, however, showed the

presence of X2 as the major hydrolysis product followed by xylotetraose (X4) and iso-

X4 putatively identified as aldotetrauronic acid a characteristic property o f xylanase

(GH10). Similarly the differences in the hydrolysis product profile obtained with

arabinoxylans (OSX, RAX and WAX) were observed. The hydrolysis product profile

obtained by TLC was confirmed by HPLC.

6.7 Purification and characterization of acetyl esterase from Malbranchea Jlava An acetyl esterase with a molecular mass and pi of 42 kDa and 3.0, respectively

was purified to homogeneity using hydrophobic interaction chromatography, chromato-

focusing and ion exchange chromatography. The purified esterase showed specific

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Summary

activity o f 17.6 pm ol min*1-mg protein*1 w ith 10.11 fold purification. The purified

esterase w as optim ally active a t S0°C and a t pH 6.0 and appreciably stable a t 60°C at

pH 6.0 and 7.0 fo r 4 h . A t 70°C , ~ 80% o f activity w as still recoverable after 4 h o f

incubation. T he activity o f purified esterase w as positively m odulated by all

m onovalent, divalent m etal ions as w ell a s SDS, PM SF, DTT and m ercaptoethanol. The

purified esterase w as m ost active against pN P acetate and it could also recognise pNP

butyrate bu t show ed very low activity against pN P ferulate. T he estim ated K„, Vmax and

kcat values fo r the purified esterase w ith pN P acetate as a substrate w ere 7.42 m M , 2.78

pm ol m in 'm g p ro te in '1 and 233.52 m in '1, respectively. T he purified esterase on the

basis o f characterization w as classified as carbohydrate esterase (CE-4).

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