High specific activity thermostable xylanase mutant at animal body temperature and its application
A technology of xylanase mutation and animal body temperature, which is applied in the field of genetic engineering, can solve the problems of loss of thermostable enzyme activity, inability to balance enzyme thermostability and enzyme activity, etc., achieve high enzyme activity and shorten the transformation of enzymatic properties The effect of improving time and heat stability
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Embodiment 1
[0036] Example 1 Cloning of genes encoding high specific activity xylanase mutants
[0037] Taking the high-temperature xylanase gene GtXyn10 from the GH10 family as the male parent, mutation primers were designed at the loop region of xylanase, and the over-lap PCR method was used to amplify the high catalytic efficiency xylanase mutant encoding gene SEQ. ID NO. 1 (GtXyn10-G92C), SEQ ID NO. 2 (GtXyn10-Q99S), SEQ ID NO. 3 (GtXyn10-G92C / Q99S), mutagenesis method and cloning method references (You, et al., 2016).
[0038] The primer sequences used are shown in Table 1:
[0039] Table 1 Primer Synthesis List
[0040]
Embodiment 2
[0041]Example 2 Preparation of high specific activity xylanase mutant
[0042] The expression vector pPIC9r was double digested (EcoR I+Not I), and the gene encoding the high specific activity xylanase mutant was double digested (EcoR I+Not I). The gene fragment of the live xylanase mutant was linked with the expression vector pPIC9r to obtain a recombinant plasmid containing the high specific activity xylanase mutant gene and transformed into Pichia GS115 to obtain a recombinant yeast strain.
[0043] Take the GS115 strain containing the recombinant plasmid, inoculate it into a 1L Erlenmeyer flask of 300mL BMGY medium, and place it at 30°C, 220rpm shaker 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 and placed again at 30°C, under the condition of 220rpm to induce the culture. 0.5 mL of methanol was added every 12 h to keep the methanol concentration ...
Embodiment 3
[0044] Example 3 Activity analysis of recombinant high specific activity xylanase mutant and wild type
[0045] 1. DNS method: The specific method is as follows: under the given pH and temperature conditions, 1 mL of reaction system includes 100 μL of enzyme solution and 900 μL of substrate, react for 10 min, add 1.5 mL of DNS to terminate the reaction, and boil in boiling water for 5 min. The OD value was measured at 540 nm after cooling. One unit of enzyme activity (U) is defined as the amount of enzyme required to decompose xylan to generate 1 μmol of reducing sugar per minute under given conditions.
[0046] 2. Characterization of recombinant high specific activity xylanase mutant and wild type
[0047] 1. The optimum pH of the recombinant high specific activity xylanase mutant and wild type was determined as follows:
[0048] The recombinant high specific activity xylanase mutant and wild type purified in Example 2 were subjected to enzymatic reaction at different pH to...
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