A linear maltooligosaccharide-producing enzyme mutant with improved maltohexaose-producing ability
A malto-oligosaccharide and maltohexaose technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low production efficiency, high production cost, difficulty in separation and purification, etc., and achieves reduction of production and processing cost, low production cost and simple process. Effect
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Embodiment 1
[0032] Example 1: Preparation of the gene sequence of the linear maltooligosaccharide generating enzyme mutant
[0033] (1) Connect the linear maltooligosaccharide generating enzyme gene whose amino acid sequence is shown in SEQ ID NO.1 (nucleotide sequence is shown in SEQ ID NO.2) to the vector pST to obtain the recombinant vector pST / mfa For the specific construction process, please refer to Section 2.2.2 on page 10 of the literature "Secretary Expression, Enzymatic Properties and Product Research of Linear Maltooligosaccharide-forming Enzyme in Bacillus subtilis".
[0034](2) Using the expression vector pST / mfa containing the target gene sequence as a template, design the complementary primer strands required for the experiment (see Table 1). The primers were synthesized by Jinweizhi Biotechnology Co., Ltd., referring to the instructions of the STAR Primer GXL kit from TaKaRa Company The indicated method performs site-directed mutagenesis. The PCR reaction system was set a...
Embodiment 2
[0038] Embodiment 2: Contain the construction of the genetic engineering bacterium of linear maltooligosaccharide producing enzyme mutant gene
[0039] The construction of the engineering bacterium containing the straight-chain maltooligosaccharide producing enzyme mutant gene is carried out according to the following method:
[0040] At 37°C, the PCR product obtained in Example 1 was treated with Dpn I for more than 2 hours, and then the treated PCR product was transformed into Escherichia coli JM109, and the transformed E.coli JM109 was coated with 100 μg / mL kana In LB agar medium containing kanamycin, cultivate overnight in a 37°C incubator for 12h, select a single colony and inoculate it into LB liquid medium containing 100 μg / mL kanamycin, at 37°C, 200r / min Cultivate overnight and extract plasmids for identification and sequencing according to the method indicated in the instruction manual of the plasmid extraction kit. The constructed target plasmid was transformed into...
Embodiment 3
[0041] Example 3: Expression of linear maltooligosaccharide generating enzyme mutants
[0042] LB medium: yeast powder 5g / L, tryptone 10g / L, NaCl 10g / L, pH 7.0.
[0043] Fermentation medium: yeast powder 30g / L, cornstarch 6g / L, KH 2 PO 4 17mM,K 2 HPO 4 72mM, pH 7.5.
[0044] (1) Host bacterium activation culture: the B.subtilis WB600 containing the expression vector plasmid pST / mfa obtained in Example 2 was streaked and separated on the LB solid medium, and placed in a 37°C constant temperature incubator for overnight culture. Take the positive single colony and inoculate it in a 250mL Erlenmeyer flask containing 50mL LB liquid medium. Add kanamycin at a final concentration of 5 μg / mL before inoculation. The Erlenmeyer flask was placed in a rotary shaker at 200r / min, and incubated at 37°C for 12h.
[0045] (2) Fermentation culture: transfer the activated seed solution to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium at an inoculum size of 4% (v / v),...
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