Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant

A maltose amylase and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as low efficiency, prolonged reaction time, and long production cycle

Inactive Publication Date: 2015-09-23
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to reduce the production of by-products, it is often necessary to prolong the reaction time to balanc

Method used

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  • Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant
  • Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant
  • Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: This example illustrates the preparation of native maltogenic amylase.

[0016] (1) Construction of maltogenic amylase recombinant bacteria

[0017] According to the amino acid sequence of maltose amylase derived from B. stearothermophilus in the NCBI database (NCBI number: 1QHO_A), the gene sequence of maltose amylase was codon-optimized according to the codon preference of Escherichia coli, and the optimized gene amyM was fully synthesized by chemical method method synthesis. The plasmid used to construct the expression vector in E. coli is pET24a(+). The pET24a(+) plasmid and the plasmid with the amyM gene were subjected to NcoI and HindIII double enzyme digestion respectively. After the digestion products were recovered by gel, they were ligated with T4 ligase overnight, and the ligated products were transformed into Escherichia coli JM109 competent cells, and the transformed products Spread on 30mg·L -1 The LB plate of kanamycin was cultured overnight...

Embodiment 2

[0021] Example 2: This example illustrates the preparation of maltogenic amylase mutants.

[0022] (1) Site-directed mutation

[0023] Based on the sequence alignment analysis of maltogenic amylase derived from B. stearothermophilus and cyclodextrin glucosyltransferase (CGTase) derived from B. circulans strain 251, the protein structure of maltogenic amylase was simulated For analysis, select the amino acid design mutation of the receptor binding site in the active center of maltose amylase. The 177th tryptophan (Trp) of maltogenic amylase was mutated into phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), aspartic acid (Asn) and serine respectively (Ser), labeled W177F, W177Y, W177L, W177N and W177S, respectively.

[0024] The site-directed mutagenesis primers for introducing the W177F mutation are:

[0025] Forward primer: 5'-GCGATATTTCTAAC TTC GACGACCGCTACGAA-3' (the underline is the mutated base)

[0026] Reverse primer: 5'-TTCGTAGCGGTCGTC GAA GTTAGAAATATCGC-3' (t...

Embodiment 3

[0042] Example 3: This example illustrates the determination of kinetic parameters of native maltogenic amylases and mutants.

[0043] Different concentrations of maltotriose solutions (0.1, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.5 mg·mL -1 ) as the reaction substrate. Take 500 μL of maltotriose solutions of different concentrations in a test tube, and preheat it in a water bath at 60°C for about 10 minutes. Add 500 μL of diluted enzyme solution sample, shake and mix, and react for 10 minutes. The reaction was terminated by adding 1 mL of 0.06N NaOH. The glucose content in the reaction system was measured with a glucose meter. The amount of enzyme required to catalyze the production of 1 μmol of glucose per minute is taken as an activity unit. The initial hydrolysis activity of maltotriose measured at different substrate concentrations was fitted using nonlinear regression in GraphPad Prism 5.0 software to obtain the K of the Michaelis-Menten equation. m and V m...

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Abstract

The invention relates to a maltogenic amylase mutant with low conversion byproducts and a production method of the maltogenic amylase mutant and belongs to the fields of genetic engineering and enzyme engineering. The production method includes the amino acid residues on catalytic activity center receptor binding sites of thermophilic fat Bacillus stearothermophilus-sourced maltogenic amylase are replaced. Compared with natural maltogenic amylase, the maltogenic amylase mutant has the advantages that the maltogenic amylase mutant is high in conversion efficiency and low in conversion byproducts during high-maltose syrup preparation. The B. stearothermophilus maltogenic amylase mutant has at least one of the following advantages that preparation cycle is shortened; byproduct proportion is lowered. The maltogenic amylase mutant is more suitable for high-maltose syrup preparation as compared with the natural maltogenic amylase.

Description

technical field [0001] The invention relates to a mutant and a mutation method of maltose amylase with low conversion by-products, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Maltose is a reducing disaccharide composed of two glucose units connected by α-1,4 glycosidic bonds, and its chemical name is 4-O-D-hexacyclic glucosyl-D-hexacyclic glucose. Its sweetness is mild, and because of its low viscosity, low hygroscopicity and good thermal stability, it can be used as a food sweetener to replace glucose and sucrose, and has great application potential in the food industry. High maltose syrup is a syrup that is mainly maltose (more than 45% maltose). One of the uses in the food industry is to make pastries, confectionery and other products. The cooking temperature of syrup is much higher than that of caramel, generally exceeding 140°C. If the maltose content is greater than 70%, or even as high as 90%, it is called ul...

Claims

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Application Information

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IPC IPC(8): C12N9/28C12N15/56C12N15/10
CPCC12N9/2417C12Y302/01133
Inventor 吴敬段绪果孙烨橙王蕾
Owner JIANGNAN UNIV
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