High thermostability β-glucosidase mutant and its application
A glucosidase and thermostability technology, which is applied in the field of high thermostability beta-glucosidase mutants, and can solve the problems of poor thermostability of beta-glucosidase and the like
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Embodiment 1
[0027] Example 1β- glucosidase random mutation library construction
[0028] 1 to carry the wild-type β- glucosidase gene pET 28a (+) - tac plasmid (pET 28a (+) - tac-Ks5A7) as a template, low mutation rate (3mutations / kb) of the gene error prone PCR (epPCR), with reference to II Random Mutagenesis kit instructions using random mutations.
[0029] Followed by two rounds of mutation:
[0030] The first round mutant template is pET 28a (+) - tac-Ks5A7,
[0031] The second round of mutation is obtained after the first round mutant plasmid pET 28a (+) - tac-2R1.
[0032] 2, in particular the primers epPCR reaction system as shown in Table 1, are shown in Table 2 epPCR:
[0033] Table 1 Construction of random mutation β- glucosidase library primer (primer epPCR)
[0034]
[0035]
[0036] Table 2 epPCR reaction system constructed β- glucosidase random mutation library
[0037]
[0038] Reaction conditions epPCR as follows: The first stage: denaturation at 95 deg.] C 2min; seco...
Embodiment 2
[0047] Example 2 Screening of glucosidase random mutations
[0048] 1. The mutant library obtained in Example 1 was applied to the LB medium plate (add 50 μg / ml kanamycin and 20 μmiptg), and then cultured overnight at a constant temperature incubator at 37 ° C, and then cultured 1d at room temperature to ensure Highly expressed expression of β-glucosidase. High temperature treatment plate colonies in the plates to kill the cells and inactive β-glucosidase in which thermal stability did not increase (first round mutation: 70 ° C for 40 min; second wheel mutation: 85 ° C treatment for 40 min). The screening indicator is a potassium phosphate solution containing 1 g / l seven leaflide, 2.5 g / L of citrate and 5 g / l agar, and carefully pour 10 ml of screening indicator to the heat treated colony. After incubation for 10 min at room temperature, the positive colonies were identified based on the formation of black halo around the colony. Use aseptic teeth to take out positive colo...
Embodiment 3
[0050] Example 3: Expression, purification of glucosidase mutants
[0051] 1. Expression of β-glucosidase mutants
[0052] The plasmids of the mutation in Example 2 were sequenced to E. coli BL21 (DE3), and the monomida was colored in the LB liquid medium at 37 ° C, 200 rpm, and cultured overnight. The ratio of 1: 100 is transferred to the LB liquid medium, oscillated into the bacterial density OD at 37 ° C, 200 rpm. 600nm At 0.85, an IPTG having a final concentration of 0.8 mm was added. 12h was continued at 25 ° C, 200 rpm, and centrifuged for 5 min, and the bacteria were collected by 10000 × g.
[0053] 2, β-glucosidase mutant purification
[0054] The β-glucosidase mutant contains a C-terminal 6 × His label, which uses NovaGen's The Purification Kit is purified, and the specific steps are as follows:
[0055] (1) Take 100ml induced culture, centrifuge, remove the supernatant, add 15 ml of pre-cooling 1 × Binding buffer, mix evenly, use ultrasonic broken bacteria, centrifuge f...
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