Sequence Analysis, Molecular Evolution And Molecular Modeling Of Xylanase

Xylanase breaks down β-1,4-linked xylopyranose chain of xylan, one of the most abundantpolysaccharide fibers in nature, and is important in the recycling of energy. The enzyme iswidely used in biotechnology. Many xylanases had been cloned and analyzed. However, theworking conditions are extreme, which often restrict the usage of xylanase. Besides to screenmicroorganism for xylanases that fit the requirement of biotechnology, it is also hoped thatprotein engineering can tailor xylanase to meet demands of industry. During the process ofprotein engineering, it is needed to understand the knowledge of sequence, function andstructure of xylanase. In the thesis, computational method was used to analyze the proteinsequence of xylanase. Construction of the data set of xylanase, protein sequence of xylanase was collected fromSwiss-prot, and the related optimum temperature and pH of each xylanase was searched forrespective xylanase. In order to find the spatial sites of the related amino acids, crystalstructures of the known structures of xylanase were collected from PDB. The relationship was analyzed between the sequence and xylanase optimal temperature. InF/10 xylanase, the di-peptides positively correlated with the optimal temperature are: GH,TD,WY;the negatively correlated ones are: GA,IA,FH,LH,NH,SR. Seen from theknown F/10 xylanase crystal structures, most of the amino acids correlated are found inα-helix area, the surface of xylanase to contact with the solution;other amino acids are inregions connecting α-helix and β-sheet, which affect the conformation of protein. These areoften connected with the stability of protein, and connected with the binding of substrate. InG/11 xylanase, the di-peptides positively correlated with the optimal temperature are: LA,LG,CD,GD,RY,CH;the negatively ones are: DC,YP,YI,CP. Seen from the knownG/11 xylanase crystal structures, most of the amino acids correlated are in the turn regions ofβ-sheet, the xylanase surface area. These regions affect the conformation of protein, the mostflexible areas in G/11 xylanase, and are easily affected by solution. The relationship was analyzed between sequence and the optimal pH of xylanase. In F/10xylanase, the di-peptides positively correlated with the optimal pH are: HQ,RQ,DL,SL,NQ;the negatively correlated ones are: GQ,PE,VE,TL,AE. Analyzed with the known F/10xylanase crystal structures, most of the related amino acids are found in the connecting areabetween α-helix and β-sheet;some are in α-helix, some are also found in cellulose bindingdomain. There are no amino acids correlated found in β-sheet areas. In G/11 xylanase, thedi-peptides positively correlated with optimal pH are: SY,GR,MR,KR, the negatively onesare YS. The amino acids correlated are found in the "Ser/Thr" area of G/11 xylanase, someare in the outer surface, others in the inner surface. The results explain the successfulimprovement of pH by introducing arginines into the xylanase surface area.Principle component analysis method was used to analyze the protein sequence of xylanase,and principle components are used to construct a discriminating function between these twofamilies. The function can be used to separate xylanase fairly well, and the principlecomponents are given meanings as secondary structures in protein.Molecular evolution of xylanase was analyzed;the evolution route is found from bacteriato epiphyte in F/10 xylanase. Compared with F/10 xylanase, G/11 xylanase seems to have arelative short time of evolution, because it only expanded in a few kinds of microorganisms.The xylanase P29126 makes a bridge between the two families. Analyzed with the differentfunction and products of the two families, it is proposed the evolution trend of xylanase isspecific of substrate and diverge-form of function. The result clearly explains the low specificand the phenomenon of monosaccharide in the products of F/10 xylanase.The structure of xylanase B from Thermotoga maritima was modeled, and the relationshipbetween the content of secondary structure and the optimum temperature in F/10 xylanase isanalyzed. Optimal temperature is found positively correlated with the content of α-helix, thecoefficient was 0.688;the content of β-sheet is also positively related, the coefficient is0.571;The content of coil is negatively related, the coefficient is -0.754. The result gives anevidence of the relationship between content of secondary structure with the stability ofprotein.