Molecular And Enzymatic Characterization Of A Neutral Xylanase And An Endoglucanase From Volvariella Volvacea

Volvariella volvacea, the edible straw mush room, is grown on an industrial scale in manytropical and subtropical regions and currently ranks sixth among cultivated mushrooms in termsof annual world wide production. It is a high-temperature basidiomycete and grow on straw assubstrate. These characteristics indicate that V. volvacea has a whole series of cellulolyticenzymes which may have better stability and efficiency, and these enzymes may be to the newpotential resource for biorefinery. Therefore, the exploitation and characterization studies ofstraw mushroom cellulase and hemicellulase have important significance.Xylanase （EC3.2.1.8） is one of the most important hemicellulase, it can hydrolyzeβ-1,4-glycosidic linkages of the xylan backbone to produce xylooligosaccharides and D-xylose.Hence, it is the crucial enzyme component of xylanolytic enzyme systems. It broadly exists inmicroorganisms and has widely commercial application in industrial processes, such as feed,paper, foodstuff, medicine and energy industries.Base on the information of an EST sequencefrom the subtractive cDNA library of Volvariella volvacea constructed before, the XynIIfull-length cDNA was amplified by RACE method. The nucleotide sequence of the xynII cDNAwere analyzed and submitted to GenBank with accession No. KC492049. The amino acidsequence had higher similarity with those of GH10family xylanases. Unlike other modularxylanases, it consists of only a single GH10catalytic domain with a unique extension （W-R-W-F）and phenylalanine and proline rich motif （T-P-F-P-P-F） at C-and N-terminus respectively,indicating it is a novel GH10xylanase. Compared with other GH10xylanase, the highestsimilarity was up to68%with Coprinopsis cinerea okayama7#130（Genebank accession No.XP₀01838887.1）XynII cDNA fragment encoding mature peptide was inserted into the plasmid pPICZ B andexpressed in Pichia pastoris KM71H （Muts, Arg+）. XynII exhibited optimal activity at pH7and60℃and stability over broad range of pH4.0-10.0. XynII showed the highest activity onbeechwood xylan, followed by soluble and insoluble oat spelt xylan, and birchwood xylan. Thespecific activities are67.27U/mg,64.27U/mg,59.17U/mg and37.94U/mg respectively. XynIIdisplayed extreme SDS resistance, retaining101.98%,92.99%and69.84%activity at thepresence of300mM SDS on birchwood, soluble oat spelt and beechwood xylan respectively. Itremained largely intact after24h of incubation with proteinase K at a protease to protein ratio of1:50at37℃. XynII was a true endo-acting xylanase lack of cellulase activity. It has weakactivity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger thanxylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase （AXE1） wasobserved for de-starched wheat bran. The highest degree of synergy （DS1.42） was obtained insequential reactions with AXE1digestion preceding XynII. In order to analysis the effect of the N-terminal and C-terminal unique extension on thecharacter of XynII, five motif deletion derivatives of XynII were constructed: XynIIΔN（N-terminal deletion mutant）, XynIIΔC4, XynIIΔC6, XynIIΔC7（4,6,7amino acids ofC-terminal deletion mutants）, and XynIIΔNC4（N-terminal and4amino acids of C-terminaldeletion mutant） were constructed. All the derivatives have the same optimal pH and temperatureexcept the optimal temperature of XynIIΔC7is55℃, but the half-life increased at55℃thanXynII except XynIIΔC7. The T1/2of XynIIΔNC4was3fold than native protein. The fivemutants XynIIΔN, XynIIΔC4, XynIIΔC6, XynIIΔC7and XynIIΔNC4was1.34，1.49，1.30，1.23and1.78fold more active than the XynII to the beechwood xylan respectively. However, theN-terminal and C-terminal deletion decreased the stability of XynII to SDS. The half-life ofXynII at4%SDS was8.65h, XynIIΔN was2.20h and all other mutants were less than2h. Allthe derivatives of XynII remained largely intact after24h of incubation with proteinase K exceptXynIIΔC7.The mutants also increase the hydrolytic ability to X3especially for XynIIΔC4，XynIIΔC6and XynIIΔNC4. CD spectra demonstrate the structure difference of mutants. So, theresults indicate that deletion of the N-, C-terminal sequence has changed the structure of XynII.Thereby, the structure difference affects the catalytic properties of the enzyme.Processivity is a feature common to many cellobiohydrolases and is thought to be a criticalstrategy for improving the catalytic efficiency for hydrolysis of crystalline substrates.In this study,we demonstrate the processivity of a GH5endoglucanase EG1from Volvariella volvacea. Theratios of soluble to insoluble reducing sugar produced from filter paper after8and24h ofexposure to EG1were6.66and8.56, respectively, suggesting that it is a processiveendoglucanase.EG1is a modular glycoside hydrolase family5endoglucanase from V.volvaceaconsisting of an N-terminal carbohydrate-binding module （CBM1） and a catalytic domain （CD）.Three derivatives of EG1containing a core domain only or additional CBMs were constructed inorder to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1under stationary and agitated conditions. All four enzymatic forms exhibited the same mode ofaction on both soluble and insoluble cellulosic substrates with cellobiose as a main end product.An additional CBM fused at either the N or C terminus reduced specific activity toward solubleand insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantlydecreased enzyme processivity. Insertion of an additional CBM also resulted in a dramaticdecrease in processivity in enzyme-substrate reaction mixtures incubated for0.5h, but this effectwas reversed when reactions were allowed to proceed for longer periods （24h）. Furthersignificant differences were observed in the substrate adsorption/desorption patterns of EG1andenzyme derivatives equipped with an additional CBM under agitated reaction conditions. Anadditional family1CBM improved EG1processivity on insoluble cellulose under highly agitatedconditions. Our data indicate a strong link between high adsorption levels and low desorptionlevels in the processivity of EG1and possibly other processive endoglucanses.