Study On Thermo-tolerant Lignocellusic Hydrolases

Here we present the relative research of Cel12B from hyperthermophilic Thermotogamaritima ATCC43589on heat tolerant mechanism, catalytic mechanism and Cel12B-CBDfusion protein. We also present the cloning, expression and characterization study on thethermostalbe xylanase A/B, β-xylosidase, β-manosidase and endoarabinanase from Thermotogathermarum DSM5069. The detailed results were as follows:（1） Structure alignment of the19hyperthermophilic endoglucanases proteins revealed thatGly30, Pro63, Pro83, Trp115, Glu131, Met133, Trp135, Trp175, Gly227and Glu229areconserved amino acid residues. In addition, the average percentage composition of residuescysteine and histidine of19endoglucanases are only0.28and0.74. It can be inferred from thenodes that there is a close relationship among the19protein from hyperthermophilic bacteriaand archaea based on phylogenetic analysis. Among these conserved amino acid residues, twoGlu residues might be the catalytic nucleophile and proton donor, Gly30, Pro63, Pro83andGly227residues might be necessary to the thermostability of protein, and Trp115, Met133,Trp135, Trp175residues are related to the binding of substrate. Reverse-PCR was used toanalyze the functional amino acid residues. It was displayed that P63K, P83K and M133Wcould decrease the enzyme activity. Therefore, theses amino acid residues are relative to thethermostability or binding of substrate.（2） When Cel12B gene fused with CBD of N-terminal of xylanases from T. maritima and T.thermarum using PCR, CBD1, CBD2, CBD3and CBD4fusion proteins were obtained. Theresults revealed that the four fusion proteins had a certain adsorption of Avcel, and CBD4showed the best performance. In addition, the optimum pH and temperature of fusion proteinswere all somewhat decreased, and became more sensitive to cations. CBD1, CBD2, CBD3andCBD4displayed some enzyme activity towards Avcel, however, the CMC enzyme activity weresignificantly reduced.（3） The xyn10B gene, encoding the endo^-1,4-β-xylanase Xyn10B from Thermotogathermarum DSM5069, was amplified from a genomic DNA. The open reading frame of thexyn10B gene was1095base pairs long and encoded the mature peptides of344amino acidswith a calculated molecular mass of40,531Da. The xyn10B gene was over-expressed inEscherichia coli. The Xyn10B was optimally active at80℃, pH6.0and the enzyme exhibiteddramatic thermostability as the residual activity maintained approximately60%of its activitywhen incubated at75℃for2h. Apparent K_m, k_catand k_cat/K_mvalues of the xylanase forbeechwood xylan were1.8mg mL^-1,519.6s^-1and288.7mL mg^-1s^-1, respectively. Confirmedby thin-layer chromatography （TLC） analysis, the end products of the hydrolysis of beechwoodxylan were mainly xylobiose, xylotriose, xylotetraose and xylopentaose but without xylose after2h hydrolysis.（4） A xylanase gene （xyn10A） with3,474bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum that encodes a protein containing1,158amino acid residues.Based on amino acid sequence homology, hydrophobic cluster and three dimensional structureanalyses, it was attested that the xylanase belongs to the glycoside hydrolase （GH） families10with five carbohydrate binding domains. When the xylanase gene was cloned and expressed inEscherichia coli BL21（DE3）, the specific enzyme activity of xylanase produced by therecombinant strain was up to145.8U mg^-1. The xylanase was optimally active at95℃, pH7.0.In addition, it exhibited high thermostability over broad range of pH4.0-8.5and temperature55-90℃upon the addition of5mM Ca2+. Confirmed by Ion Chromatography System （ICS）analysis, the end products of the hydrolysis of beechwood xylan were xylose, xylobiose,xylotriose, xylotetraose, xylopentaose and xylohexaose. The xylanase from T. thermarum is oneof the hyperthermophilic xylanases that exhibits high thermostability, and thus, is a suitablecandidate for generating XOs from cellulosic materials such as agricultural and forestryresidues for the uses as prebiotics and precursors for further preparation of furfural and otherchemicals.（5） A β-xylosidase gene （Tth xynB3） of2,322bp was cloned from the extremelythermophilic bacterium Thermotoga thermarum DSM5069that encodes a protein containing774amino acid residues, and was expressed in Escherichia coli BL21（DE3）. The phylogenetictrees of β-xylosidases were constructed using Neighbor-Joining （NJ） and Maximum-Parsimony（MP） methods. The phylogeny and amino acid analysis indicated that the Tth xynB3β-xylosidase was a novel β-xylosidase of GH3. The optimal activity of the Tth xynB3β-xylosidase was obtained at pH6.0and95℃and was stable over a pH range of5.0-7.5andexhibited2h half-life at85℃. The kinetic parametersK_mand V_maxvalues forp-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were0.27mM and223.3U/mg,0.21mM and75U/mg, respectively. The k_cat/K_mvalues forp-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were1,173.4mM^-1s^-1and505.9mM^-1s^-1, respectively. It displayed high tolerance to xylose, with Kivalueapproximately1000mM. It was stimulated by xylose at higher concentration up to500mM,above which the enzyme activity of Tth xynB3β-xylosidase was gradually decreased. However,it still remained approximately50%of its original activity even if the concentration of xylosewas as high as1000mM. It was also discovered that the Tth xynB3β-xylosidase exhibited ahigh hydrolytic activity on xylooligosaccharides. When5%substrate was incubated with0.3UTth xynB3β-xylosidase in200μL reaction system for3h, almost all the substrate wasbiodegraded into xylose. The article provides a useful and novel β-xylosidase displayingextraordinary and desirable properties: high xylose tolerance and catalytic activity attemperatures above75℃, thermostable and excellent hydrolytic activity onxylooligosaccharides.（6） A β-mannosidase gene （Tth man5） of1,827bp was cloned from the extremelythermophilic bacterium Thermotoga thermarum DSM5069that encodes a protein containing608amino acid residues, and was over-expressed in Escherichia coli BL21（DE3）. The resultsof phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5is a novel β-mannosidase of glycoside hydrolase family5. The optimal activity of the TthMan5β-mannosidase was obtained at pH5.5and85℃and was stable over a pH range of5.0to8.5and exhibited2h half-life at90℃. The kinetic parametersK_mand V_maxvalues forp-nitrophenyl-β-D-mannopyranoside and1,4-β-D-mannan were4.36±0.5mM and227.27±1.59μmol min^-1mg^-1,58.34±1.75mg mL^-1and285.71±10.86μmol min^-1mg^-1, respectively. Thekcat/K_mvalues for p-nitrophenyl-β-D-mannopyranoside and1,4-β-D-mannan were441.35±0.04mM^-1s^-1and41.47±1.58s^-1mg^-1mL, respectively. It displayed high tolerance to mannose, witha Kivalue of approximately900mM. This work provides a novel and useful β-mannosidasewith some desirable properties: high mannose tolerance and catalytic activity at temperaturesabove80℃, thermally stable and wonderful hydrolytic activity. Therefore, these characteristicsconstitute a powerful tool for improving the enzymatic conversion of mannan throughsynergetic action with other mannan-degrading enzymes.（7） Arabinan is an important plant polysaccharide which can be degraded mainly by twohydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, thecharacterization and application in arabinan degradation of a novel endo-arabinanase fromThermotoga thermarum were investigated. The recombinant enzyme was over-expressed inEscherichia coli BL21（DE3）, and then purified by heat treatment followed by purification on anickel affinity column. The purified endo-arabinanase was characterized with respect tosubstrate specificity and biochemical properties. The endo-arabinanase was optimally active atpH7.0and75℃. The endo-arabinanase residual activity retained more than80%of its initialactivity after being incubated at75℃or80℃for2h.The results showed that theendo-arabinanase was very effective for arabinan degradation at higher temperature. Whenlinear arabinan was used as substrate, the apparent K_mand V_maxvalues were determined to be12.3±0.15mg mL1and588.3±7.1μmol mg1min1respectively （at pH6.5,75℃）, and thecalculated kcatvalue was495.4±5.9s^-1.