40 DEG C high-specific-activity xylanase mutant and construction method and application thereof
A xylanase mutation and mutant technology, applied in the field of genetic engineering, can solve the problems of high blindness, low frequency of beneficial mutations, and difficulty in obtaining target strains, etc.
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Embodiment 1
[0064] Cloning of embodiment 1 high specific activity xylanase mutant coding gene
[0065] Using the GH10 family high-temperature xylanase gene Xyl10C derived from Bispora sp.MEY-1 as the male parent, design mutant primers in the loop region of xylanase, and use over-lap PCR to amplify xylanase with high catalytic efficiency. Carbohydrase mutant coding gene SEQ ID NO.1 (XYLΔN-M137E, 1023bp), SEQ ID NO.2 (XYLΔN-MF137 / 138DA, 1023bp), SEQ ID NO.3 (XYLΔN-MF137 / 138DS, 1023bp), SEQ ID NO. ID NO.4 (XYLΔN-MF137 / 138QP, 1023bp) and SEQ ID NO.5 (XYLΔN-MF137 / 138SL, 1023bp), mutation method and cloning method reference (You, et al., 2016).
[0066] Table 1 Gene-specific primers of high specific activity xylanase mutants at 40°C
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Embodiment 2
[0069] The preparation of embodiment 2 high specific activity xylanase mutants
[0070] The expression vector pPIC9r was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene encoding the high specific activity xylanase mutant was double enzyme digested (EcoR I+Not I), and the cut coding mature high specific activity xylanase The gene fragment of the mutant glycanase is connected with the expression vector pPIC9r to obtain a recombinant plasmid containing the gene of the mutant xylanase with high specific activity and transform into Pichia pastoris GS115 to obtain a recombinant yeast strain.
[0071] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL of 0.5% methanol for precipitation. The BMMY medium was resuspended, an...
Embodiment 3
[0076] Embodiment 3 activity analysis of recombinant high specific activity xylanase mutant and wild type
[0077] 1. DNS method: the specific method is as follows: under the given pH and temperature conditions, 1mL reaction system includes 100μL enzyme solution, 900μL substrate, react for 10min, add 1.5mLDNS to terminate the reaction, boil in water for 5min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose xylan to generate 1 μmol reducing sugar per minute under given conditions.
[0078] 2. Determination of the properties of recombinant high specific activity xylanase mutants and wild type
[0079] 1) The optimal pH determination method of recombinant high specific activity xylanase mutant and wild type is as follows:
[0080] The recombinant high specific activity xylanase mutant purified in Example 2 and the wild type were subjected to enzymatic reactions at different pHs to determin...
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