Salt-tolerant xylosidase mutant t329e and its preparation method and use
A technology of xylosidase and wild xylosidase, which is applied in the field of salt-tolerant xylosidase mutant T329E and its preparation, can solve problems such as lack of catalytic activity, and achieve the effect of enhanced stability
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experiment example 1
[0031] Construction and Transformation of Experimental Example 1 Expression Vector
[0032] According to the xylosidase nucleotide sequence KY391885 (SEQ ID NO.4) recorded in GenBank, the gene hJ14GH43 encoding the wild xylosidase HJ14GH43 was synthesized; the gene t329e (SEQ ID NO.2) encoding the mutant enzyme T329E was also synthesized.
[0033] The synthetic xylosidase nucleotide sequence and mutant enzyme T329E nucleotide sequence were respectively connected with the expression vector pEasy-E1 to obtain the expression vector containing hJ14GH43 and t329e, and the connection products were respectively transformed into Escherichia coli BL21 (DE3) to obtain the respective Recombinant strain expressing wild enzyme HJ14GH43 and mutant enzyme T329E.
experiment example 2
[0034] Preparation of Experimental Example 2 Wild Enzyme HJ14GH43 and Mutant Enzyme T329E
[0035] The recombinant strains containing hJ14GH43 and t329e were inoculated in LB (containing 100 μg mL - 1 Amp) medium, shake rapidly at 37°C for 16h.
[0036] Then, the activated bacterial solution was inoculated into fresh LB (containing 100 μg mL -1 Amp) culture medium, rapid shaking culture for about 2 ~ 3h (OD 600 After reaching 0.6-1.0), add IPTG at a final concentration of 0.1 mM for induction, and continue shaking culture at 20° C. for about 20 h.
[0037] Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the cells with an appropriate amount of pH7.0 Tris-HCl buffer solution, the cells were ultrasonically disrupted in a low-temperature water bath.
[0038] After the crude enzyme solution concentrated in the cells was centrifuged at 12,000rpm for 10min, the supernatant was aspirated and the target protein was affinity and eluted with Nickel-NTAAgaro...
experiment example 3
[0040] Determination of the properties of wild enzyme HJ14GH43 and mutant enzyme T329E purified in Experimental Example 3
[0041] The activities of the purified wild enzyme HJ14GH43 and the mutant enzyme T329E were determined by the pNP method, as follows:
[0042] Dissolve pNPX in the buffer solution to make the final concentration 2mM; the reaction system contains 50μL of appropriate enzyme solution and 450μL of 2mM substrate; after the substrate is preheated at the reaction temperature for 5min, add the enzyme solution and react for an appropriate time, then add 2mL 1M Na 2 CO 3 The reaction was terminated, and the released pNP was measured at a wavelength of 405 nm after cooling to room temperature; 1 enzyme activity unit (U) was defined as the amount of enzyme required to decompose the substrate to produce 1 μmol pNP per minute.
[0043] 1. Stability of purified wild enzyme HJ14GH43 and mutant enzyme T329E in NaCl
[0044] The purified enzyme solution was placed in 3....
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