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Alpha-glucosidase mutant and application thereof

A glucosidase and mutant technology, applied in the field of genetic engineering, can solve the problems of unfavorable environmental protection, complicated operation, severe reaction conditions and the like

Active Publication Date: 2019-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, the resistant content of pyrodextrin after high-temperature acid and heat hydrolysis of starch can reach 50%. In industrial production, after treatment with glucoamylase, it can form a finished product with more than 80% of resistant components. However, this production method has severe reaction conditions and Complicated operation, not conducive to environmental protection

Method used

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  • Alpha-glucosidase mutant and application thereof
  • Alpha-glucosidase mutant and application thereof
  • Alpha-glucosidase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Cloning of α-glucosidase gene derived from Aspergillus nidulans and construction of expression vector

[0046] The synthetic target gene AgdB(-) (nucleotide sequence shown in SEQ ID NO.2) was connected to the cloning vector pMD19T, called pMD19T-AgdB(-), transformed into E.coli JM109 for further cloning, and the target gene was digested and recovered. The gene was connected with the expression vector pPIC9K overnight at 16°C, transformed into E.coli JM109, coated with LB plate containing kana (30μg / mL) resistance, cultured at 37°C for 10-12h, picking transformants, extracting recombinant plasmids and Double digestion verification, and then determine the DNA sequence of the verified recombinant plasmid, the positive clone is AgdB(-)-pPIC9K.

Embodiment 2

[0047] Embodiment 2: Transformation of recombinant plasmid AdgB(-)-pPIC9K

[0048] The plasmid AdgB(-)-pPIC9K was electrotransferred into the competent Pichia pastoris KM71 prepared in advance to obtain the genetically engineered bacteria Pichia pastoris KM71 / AgdB(-)-pPIC9K, which was cultured on an MD plate at 30°C for 3-4 days. After the transformants were obtained, they were picked on G418 / YPD plates with different concentrations to screen positive clones, and cultured at 30°C for 24h. Pick a single colony into 10mL of YPD liquid medium, culture at 30°C for 24h, save the glycerol tube, and store it in a -80°C refrigerator. After the verification is correct, shake flask fermentation is carried out to produce enzyme.

Embodiment 3

[0049] Embodiment 3: Shake flask fermentation produces enzyme

[0050] The recombinant Pichia strain obtained in Example 2 was inoculated in YPD medium, and after culturing at 30°C for 24h, it was transferred to 50mL BMGY medium with 5% inoculum size and cultivated in a constant temperature shaker at 30°C for 24h, and then Centrifuge at 5000rpm for 5min, discard the supernatant, collect the bacteria, hang the bacteria with 25mL BMMY medium, add 1.5% methanol to induce enzyme production in the bacteria, cultivate in a constant temperature shaker at 30°C for 120h, add 1.5% methanol every 24h After the induced fermentation, centrifuge at 5000rpm for 20min, and the supernatant is the recombinant α-glucosidase. The measured activity of α-glucosidase in the supernatant can reach 16U / mL, which is 1.58 times higher than that of the wild enzyme. Shake flask fermentation to OD 600 The protein content of 40 is 0.4g / L. The results of protein electrophoresis showed that there was a band...

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Abstract

The invention relates to an alpha-glucosidase mutant and application thereof, in particular to an alpha-glucosidase mutant and application of the alpha-glucosidase mutant to production of isomaltooligosaccharide and resistant dextrin, and belongs to the technical field of gene engineering. An alpha-glucosidase mutant gene from aspergillus nidulans is transferred into pichia pastoris to be expressed; the recombinant alpha-glucosidase is used for preparing isomaltooligosaccharide and resistant dextrin; under the conditions of the temperature being 45 DEG C, the pH being 5.5, the enzyme adding quantity being 5 U / g and the substrate concentration being 300 g / L, the yield of the isomaltooligosaccharide reaches 216 g / L; the conversion rate is 72 percent. When pyrodextrin is used as a substrate,three enzymes of branching enzymes, alpha-CGT enzymes and alpha-glucosidase are compounded to improve the ingredients of resistant dextrin; the resistant ingredients of the pyrodextrin can be improvedto 70 percent from the original 44 percent. By the method, the production efficiency is effectively improved; the production cost is reduced.

Description

technical field [0001] The invention relates to an alpha-glucosidase mutant and its application, in particular to an alpha-glucosidase mutant and its application in the production of isomaltose oligosaccharides and resistant dextrin, belonging to the technical field of genetic engineering. Background technique [0002] Alpha-glucosidases have industrial applications in the production of isomaltose oligosaccharides (IMOs). Isomalt oligosaccharide, also known as branched oligosaccharide, is a syrup produced by whole enzymatic process using starch as raw material. It consists of 2-10 glucose residues and has at least one α-1,6 glycosidic bond in the molecule. , the others are α-1,4 glycosidic bonds. Isomalt oligosaccharide is a product with high production and sales volume among functional oligosaccharides in the world. It mainly promotes the proliferation of beneficial bacteria in the human intestine, inhibits the growth of many pathogenic bacteria and spoilage bacteria, enha...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N15/56C12N1/19C12P19/14C12P19/04C12P19/02C12P19/12C12R1/84
CPCC12N9/2408C12P19/02C12P19/04C12P19/12C12P19/14C12Y302/0102
Inventor 陈晟吴敬任莉琼
Owner JIANGNAN UNIV
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