| Xylanase is compound hydrolases composed of endonuclease and excision enzyme. It candegrade xylan into xylo-oligosaccharide or xylose, and has abroad application potential onfood,papermaking,feeding and other industrial fields. Xylanase comes from animal,plantsand microorganism, and xylanase which comes from microorganism is most widely used.However, at present, the enzyme activity of xylanase is low, which cannot fulfill the industrialdemand, so as to restrict its application in industrial fields. The xylan is digested intoxylo-oligosaccharides and oligosaccharides by xylanase, which can be digestion andabsorption by animal. As feeding enzyme, the main problem of xylanase is the temperaturetoo high during processing causing the activity reduced. The research shows that thepelletization temperature of chicken feed is65-75℃, pig feed is about70-80℃, the aquaticfeed is about85-90℃and the natural xylanase optimum temperature is50-60℃which haveless tolerance to extreme environments.Therefore, it has a great significance of feed industry by getting xylanase which have highactivity and heat resistance. Protein engineering of the thermostable xylanase xylBYG wasperformed to obtain enzymes with improved thermal tolerance.In this study, xylanase gene (xynA) from an alkali resistant bacillus pumilus (BYG5-20N)(previous selected by our laboratory) was used as the first round of epPCR template. For thesecond round of epPCR, two beneficial mutations from the first round of epPCR as a template.xynA was amplified from bacillus pumilus genome, and then cloned into pGEM-T vector, wegot the needed xylanase gene.xynA by digested pGEM-xynA with EcoR I and Sac I. We got the vector backbone bydigested pET-32a (+) with EcoR I and Sac I. These two fragments were linked together thenpETX yield. Then which was transformed into E. coli BL21competent cells, Coating in LBplate, and cultured for12h at37℃to construction of the mutant library.High temperature resistant mutants were sequenced by the identification of evacuation ofHealth Biological Engineering Company. Compared with the wild-type gene, to findhomologous sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/) and to compareand select the template of structural simulation results. Input the amino acid sequence of the target protein and the template PDB to SWISS–MODEL (http://swissmodel.expasy.org/), andthen it generated simulation structure, which can analysis the mutation effects.The results showed that two mutant02C05and13A09were obtained from the first roundof epPCR which had retained40%activity after the heat treatment experiments. It had fouramino acid substitutions T52I, G112D, M54I and A98V in the first round of screening. A newmutant37D07was appeared in the second round of epPCR which had four amino acidsubstitutions E120D, A98V, T52I, G112D. Only one mutant23E06inherited from the firstround of epPCR amino acid substitution.Analyze the thermostability by measuring the residual activity between wild-typexylanase xylBYG and the mutant xylanase. When the temperature reaches60℃, the wild-typexylanase xylBYG had lost90%relative enzyme activity, also retained80%relative enzymeactivity in the mutant enzyme23E06and37D07, mutant enzyme02C05and13A09from thefirst round of epPCR retained50%relative enzyme activity.Determine the enzyme optimum temperature by measuring the relative activity. Theoptimum temperature of wild-type xylanase xylBYG was55℃, mutant02C05,13A09fromthe first round of epPCR was63℃, mutant37D07was68℃. The most thermostable mutantenzyme37D07contained4amino acid substitutions. Its reaction temperature for maximumactivity increased from55℃to68℃, and relative activity increased by17%. The mutant isof potential interest for industrial applications. |
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