A kind of saccharification enzyme mutant and its application
A mutant and glucoamylase technology, applied in the fields of genetic engineering and enzyme engineering, to improve product purity and yield, reduce residual sugar content, and prevent the formation of non-fermentable sugars
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Embodiment 1
[0073] Example 1 Construction of glycoamylase mutant vector expression vector
[0074] The amino acid sequence of the wild-type glycoamylase gene is shown in SEQ ID NO:1. Through the homology alignment in the PDB data, the sequence and protein structure of saccharification enzymes with high similarity were obtained, and the SWISS-MOLDEL online modeling website was used to conduct homology modeling to screen out the saccharification enzymes that may affect the transglycosidic activity of saccharification enzymes. mutant sites, and designed single and combined mutants M1-M23 (see Table 2 for the corresponding mutant identifiers). According to the design, it was first sent to Suzhou Hongxun Biotechnology Co., Ltd. to synthesize the mutant gene M5 with 4 combined mutation sites, and then the M5 gene was used as a template to design the primers for the mutation sites according to the mutation sites, and the overlap extension PCR was used to obtain 3 Combination mutants M1 and M2 o...
Embodiment 2
[0077] Example 2 Construction of Pichia pastoris recombinant strain
[0078] The recombinant plasmids pJ912-WT and pJ912-M1~pJ912-M23 were linearized with SacI restriction endonuclease and recovered by electrophoresis respectively. The plasmids recovered after linearization were electrotransformed into Pichia pastoris X33 host strain, and spread to the host strain containing 400 μg Cultured on YPD plates of / mL Zeocin at 30°C for 2-3 days. Pichia pastoris positive transformants were identified by PCR, and the correct positive transformants identified by PCR defined corresponding recombinant strains such as X33WT, X33 / M1, X33 / M2, ...... X33 / M23.
Embodiment 3
[0079] Example 3 Induction of expression of wild-type and mutant glycoamylase genes.
[0080] Eight recombinant strains of Pichia pastoris containing wild-type and different mutant saccharification enzyme genes were respectively inoculated into 1.5mL BMD1 medium (0.2M potassium phosphate, 13.4g / L YNB, 0.4mg / L biotin, 1.1% glucose) ) in a 48-well deep-well plate at 28°C for 48-60h. Then add 1.25mL BMM2 (0.2M potassium phosphate, 13.4g / L YNB, 0.4mg / L biotin, 1% methanol), 28°C, 220rpm shaking culture for 12h, and then add 250μL BMM10 (0.2M potassium phosphate, 250μL BMM10 every 24h, 13.4g / LYNB, 0.4mg / L biotin, 5% methanol) for methanol induction, 28°C, 220rpm shaking culture for 72h. The fermentation broth was collected upon expiration, centrifuged at 12,000 r / min for 10 min, the bacteria were removed, and the supernatant was used for enzyme activity assay.
[0081] For the wild-type and each mutant recombinant strains, 3 strains with higher enzyme activity were selected and i...
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