The Improvement For Salt Tolerance Of Endo-xylanase And Xylosidase

Endo-xylanase and ?-xylosidase are very important glycoside hydrolases,they have important application value in the food,feed,papermaking,textile,energy and other industries.Salt-tolerant enzymes are active and stable at high salt concentrations and can be used in high salt food processing,seafood processing,washing and other biotechnology fields with high salt concentrations.High salt environments can also prevent microbial contamination,avoiding sterilization and other energy consumption.Through the genetic engineering methods,heat,acid,alkali and other properties of enzymes can be modified in order to make them adapt to various applications.However,the modification of salt tolerance of enzymes is rarely reported.In this paper,the salt tolerance of glycosyl hydrolase family 10 endo-xylanase and family 43 xylosidase were improved.The results are as follows:1.The improvement of salt tolerance for endo-xylanaseThe mutant s42f11 and s45f07 were used as materials obtained by endo-xylanase gene family rearrangement in the previous study of our laboratory.Their enzymatic properties were determined.Both mutants are active at high temperatures.The optimum temperatures of s42f11 and s45f07 were both 80 oC,while the optimum temperatures of the wild-type Xyn AGN16 L and XynAHJ13 were 50 and 70 oC,respectively.With the addition of 20%(w/v)NaCl,s42f11 and s45f07 had more than 46% and 15% activities compared to the wild type enzymes,respctively.With the addition of 20%(w/v)Na2SO4,s42f11 and s45f07 had more than 10% and 20% activities compared to the wild type enzymes,respctively.However,the two mutants had similar properties with the wild type enzymes when KCl,KNO3,NaNO3 and(NH4)2SO4 were added to the reaction system.2.The improvement of salt tolerance for xylosidase HJ14GH43The xylosidase gene HJ14GH43 provided by the laboratory was used.After analyzing the homologous sequence,and the mutation at position 322 was changed to aspartic acid by using the method of site-directed mutagenesis to obtain mutant HJ14GH43 V322 D.And the mutant was successfully heterologously expressed in E.coli.Its enzymological properties were determined.The optimal reaction temperature of the mutant HJ14GH43 V322 D is 25 oC.At 20 oC for 1 h,the enzyme activity retained more than 70% activity.In the reaction system containing 20%(w/v)NaCl,NaNO3 or(NH4)2SO4,the activity of mutant V322 D increased by 20% over the wild type HJ14GH43.In the reaction system containing 20%(w/v)NaCl,Na2SO4 or(NH4)2SO4 for 1 h,the salt-stability of the mutant was once to twice as accurately as the wild type.The activities of mutants in KCl and KNO3 were not significantly changed compared with the wild type enzyme.In conclusion,two novel endo-xylanase mutants,s42f11 and s45f07,were obtained by molecular techniques.The basic enzymatic properties and six salts tolerance were studied.Moreover,the salt-tolerant xylosidase mutant HJ14GH43 V322 D was obtained by site-directed mutagenesis,and its enzymatic properties were studied.This study will contribute to the application of salt-tolerant xylanases and salt-tolerant mechanisms.