Study On The Mutations Of An Extreme Thermostable Xylanase 1VBR By Error-prone PCR

Xylan is the main component of hemicellulose,and its content is only lower than cellulose in nature.Therefore,xylan is a very important renewable resource,and it is mainly used in industry to produce xylo-oligosaccharides.Xylanases??-1,4-xylanase?can specifically hydrolyze?-1,4 glycosidic bond of xylan and decompose xylan into xylo-oligosaccharides and a small amount of xylose.Xylanases are widely distributed among nature and are the most important type of enzyme in xylan-degrading enzymes system.Among them,heat-resistant xylanases are a kind of glycoside hydrolases that are widely used in many industrial fields including pulp bleaching,food industry,biodegradation and biofuel production.Therefore,a xylanase gene,1VBR derived from Thermotoga maritima MSB8 and conserved in the laboratory,was used as study object after codon-optimized.Genes after codon-optimized have a gene accession number of KR078269 in GenBank.In this study,we extracted xylan from two kinds of materials,corn cobs and poplar bark,by alkali extraction.The experiment verified the extraction contain sufficient amount of xylan and contain almost no monosaccharide,which proved that the extraction can be used as a xylan substrate in the xylanase enzymatic assay.Then,the xylanase gene 1VBR was treated by error-prone PCR to obtain a series of mutants.The error-prone PCR was partially modified on the basis of ordinary PCR technology.The DNA polymerase was rTaq enzyme which can lower the correct rate of PCR.And the concentration of Mg²⁺and Mn²⁺in the PCR system was changed to ensure that the PCR could amplify the target fragment,while generating as many mutant copies as possible.A total of 121 mutants were obtained by 6 batches of error-prone PCR,12mutant strains were screened out and their relative enzyme properties were determined.Among the 12 mutant strains,the optimal temperatures for most of the mutants were still90°C-100°C.The optimum temperature for 2-6 varied greatly and was down to 70°C,there was more than 50%relative enzyme activity between 55°C and 85°C and there was still more than 20%relative enzyme activity between 40°C and 90°C.As the temperature decreased,the relative enzyme activity of 2-5 decreased significantly slower than other mutants and wild-type.The optimal temperature of 2-5 was 90°C,there was100%relative enzyme activity at 90°C while the relative enzyme activity at 50°C was still around 50%,indicating 2-5 was a wide temperature range mutant.The most suitable pH values changed little,were still between pH 5.5–6.0.The relative enzyme activities was lower than 20%when the pH values were decreased to 4.5.Likewise,when the pH value exceeded 8.0,there was about 30%relative enzyme activity in addition to 2-6,and the relative enzymatic activities of the other mutants and wild type was lower than 20%.In terms of thermal stability,the thermal stabilities of 1-12 and 2-6 at their respective optimum temperature conditions were significantly better than that of the wild type,while the thermal stability of 2-5 at the optimum temperature is similar to that of the wild type.6-3,6-4 and 3-5 have the worst thermal stabilities,indicating that one or a few mutation sites destroyed their thermal stabilities.There were varying degrees of thermal stabilities reduction phenomenon.Finally,for pH stabilities,3-5,6-2 were similar to wild type,which were more stable in the pH range of 7.0–10.0,2-5 was more unstable in the neutral range,6-4 was unstable in the alkaline range.Overall there was no significant change in pH stabilities,either wild-type or mutant tolerate pH.In addition,all 12 mutants were sequenced,and the sequence results were analyzed by Vetcor NTI software to determine the DNA and amino acid mutation sites.The PDB software was then used to perform some analysis of the relationship between the properties of the enzymes and the mutations,but the specific site was kept confidential as needed.Finally,three mutant strains on obvious changes were chosen,namely 1-12,2-5 and2-6.The fermentation broth was concentrated and the xylanases was purified by nickel-affinity chromatography column through histidine tag attached on the carrier.After SDS-PAGE proved that almost all the other proteins were removed from the samples,Km values and specific enzyme activities were measured.1-12 is similar to the wild-type in Km value but slightly less active than wild-type,however,given that the thermal stability of 1-12 is better,further comparative studies are possible.However,the Km value of 2-5increased a lot,probably because the mutation sites destroyed its binding ability with the substrate,and its activity was much lower.The Km value of 2-6 was also increased,but did not rise as much as 2-5,and its activity is much lower too.The relationship between these dramatic changes and mutation sites deserves to be further studied.