Engineering Four Different Kinds Of GH11 Xylanases And Studying Their Catalytic Properties

As one of the glucoside hydrolases,xylanase are widely used in food and non-food industrial application.In order to meet the need of industrial production,more and more properties of xylanases were studied.This paper focused on engineering N-terminal area and studing the change of hydrolytic characteristic,mainly including five parts as follows:First,engineering a GH11 xylanase T-Xyn from Talaromyces thermophilus F1208 and studying its hydrolytic characteristic.In the work,a hybrid enzyme?T-XynFM?was created by replaceing the N-terminal extension FPTGNTTELEKRQTTP?1-16?with AFTVGNGQ and substituting amino acid Phe193 with Ser in the C-terminal.In the products of T-XynFM,almost no xylose was produced when using xylotriose?X3?,xylotetraose?X4?,and xylopentaose as substrates.Moreover,over 60%X4 existed in hydrolyzed products of X3,indicating that T-XynFM possesses formidable transglycosylation properties.When beechwood xylan,birchwood xylan and oat-spelt xylan were used as the substrates,smaller xylose was produced than that of T-Xyn,the most reduction was 90.22%with beechwood xylan.The property would attract attention in producing xylooligosaccharide.Second,engineering a GH11 xylanase Srxyn from Streptomyce rochei L10904 and studying its hydrolytic characteristic.In this part,a hybrid enzyme?SrxynFM?was created by replaceing the N-terminal extension ATTITT?1-6?with AFTVGNGQ and substituting amino acid Ile?99?with Thr in the cord.SrxynFM showed enhanced ability of producing xylobiose?X2?and xylotriose?X3?when using beechwood xylan,birchwood xylan and oat-spelt xylan as the substrates,the most increment was 41.32%and 64.29%with beechwood xylan,respectively,and no xylose was produced.The engineered xylanase displayed greater application potential in oligosaccharide preparation industry.Third,engineering two GH11 xylanases Pjxyn and Paxyn from Penicillium janthinellum MA21601 and Paenibacillus sp.39631,respectively,and studying their hydrolytic characteristics.Three mutants PjxynS?27?S?39?,PjxynS?27?S?186?and PjxynS?39?S?186?were created by introducing disulfide bridge Ser?27?Cys-Ser?39?Cys,Ser?27?Cys-Ser?186?Cys and Ser?39?Cys-Ser?186?Cys,respectively.When xylotriose?X3?was used as the substrate,it could be degraded more easily by the three mutants comparing with Pjxyn.More xylose and xylobiose?X2?were produced by mutants when the commercial xylans?beechwood xylan,birchwood xylan and oat-spelt xylan?were used as the substrates.PjxynS?27?S?39?showed the most increment with 87.62%xylose?beechwood xylan?and 69.91%X2?oat-spelt xylan?.Paxyn was engineered by introducing a disulfide bridge Asn?23?Cys-Asn?186?Cys to produce PaxynN?23?N?186?.The xylotetraose?X4?could be degraded greater by PaxynN?23?N?186?than that of Paxyn.Moreover,more xylose and X2 were produced in the hydrolysis products comparing with Paxyn.Almost no xylose was produced in the products of Paxyn.However,the most xylose was 3.30?mol ml^-1 with beechwood xylan in the products of PaxynN?23?N?186?and the most increment of X2 was 86.59%?beechwood xylan?.The mutants with disulfide bonds showed similar hydrolysis phenomenon that more xylose and X2 were produced,indicating that the disulfide bonds have an effect on the hydrolytic characteristic of xylanases.Fourth,engineering Srxyn from Streptomyce rochei L10904 and T-Xyn from Talaromyces thermophilus F1208,and studying their hydrolytic characteristics.The disulfide bond Gln?39?Cys-Asn?186?Cys was introduced in Srxyn to creat SrxynQ?39?N?186?.It keep the line with PjxynS?27?S?39?,PjxynS?27?S?186?,PjxynS?39?S?186?and PaxynN?23?N?186?that more xylose and X2 were produced comparing with Srxyn when beechwood xylan,birchwood xylan and oat-spelt xylan were used as the substrates,the most increment was 74.43%and 12.22%with oat-spelt xylan,respectively.According to the feature of T-Xyn,three mutants were constructed:T-XynC?122?C?166?was created by engineering Cys?122?Ser and Cys?166?Ser;T-XynT?38?S?50?was created by introducing a disulfide bond Thr?38?Cys-Ser?50?Cys;T-XynT?38?S?50?C?122?C?166?was produced by replacing Cys?122?and Cys?166?with Ser and introducing a disulfide bond Thr?38?Cys-Ser?50?Cys.Three mutants showed opposite results that fewer xylose and X2 were produced comparing with T-Xyn,especially T-XynT?38?S?50?and T-XynT?38?S?50?C?122?C?166?.T-XynT?38?S?50?C?122?C?166?showed the most reduction with 64.80%xylose and 31.90%X2 when using beechwood xylan as the substrate.Fifth,engineering N-terminal region of four different GH11 xylanases and studying their hydrolytic characteristics.The hydrolytic characteristics were altered when the disulfide bonds were introduced in four different kinds of GH11 xylanases,involving both of positive and negative impact.All strategies?N-terminal was substituted and disulfide bond was introduced in the N-terminal area?were applied in the N-terminal region of xylanases.Moreover,these strategies did not directly contact with a series of substrate binding sites in the catalytic cleft.However,the hydrolytic characteristics were modulated due to these engineering strategies,and the alteration were displayed in the production of xylose and xylobiose?X2?.The internal relations were explored based on the structural stability of GH11 xylanases and the alteration of hydrolysis products.The enhanced structural stability of GH11 xylanases would improve the ability to identify the substrates,on the contrary,the ability would be weakened.Thus the hydrolytic characteristics were changed,and finally it was displayed in the hydrolysis products of xylanases.The engineering strategies of N-terminal region with numerous research reports basis were employed in this work including the replacement of N-terminal and the construction of disulfide bonds,paying more attention to the hydrolytic characteristic of GH11xylanases and providing important research idea to study it.Furthermore,the effect of N-terminal engineering on the characteristics of xylanases were broaden,providing more comprehensive theoretical guidance for improving the properties of xylanases.