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Preparation and application of a kind of maltogenic amylase mutant

A maltogenic amylase and mutant technology, which is applied in the field of enzyme engineering, can solve the problems of reducing product purity and reducing the final yield of maltose, and achieves the effects of less conversion by-products, important industrial application potential, and high conversion efficiency.

Active Publication Date: 2020-11-03
湖南金悦降解塑料制品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the trisaccharides and tetrasaccharides in the product are relatively similar to maltose in nature, they often become the main impurities in the separation and purification process, which not only directly reduces the product purity, but also greatly affects the crystallinity of maltose, syrup viscosity and moisture content of the final product. Adverse effects, greatly reducing the final yield of maltose

Method used

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  • Preparation and application of a kind of maltogenic amylase mutant
  • Preparation and application of a kind of maltogenic amylase mutant
  • Preparation and application of a kind of maltogenic amylase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Preparation of wild maltogenic amylase.

[0021] (1) Construction of maltogenic amylase recombinant bacteria

[0022] According to the amyM amino acid sequence on NCBI (NCBI number: AAA22233.1), the sequence was codon-optimized, and the gene sequence amyM of maltogenic amylase was synthesized by chemical total 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 LB plates containing 100mg / L kanamycin, culture overnight at 37°C, pick 2 single colonies on the plate, insert them into LB liquid medium, extract plasmids for verification after 8 hours, the result is correct, and enric...

Embodiment 2

[0026] Embodiment 2: preparation of maltogenic amylase mutant

[0027] (1) Single mutation

[0028] The 198th lysine (Lys) in the maltose amylase was mutated into glutamic acid (Glu) and tyrosine (Tyr) respectively, marked as K198E, K198Y; the 290th asparagus in the maltose amylase Amino acid (Asp) was mutated into glycine (G, ly) and threonine (Thr) respectively, labeled as D290G and D290T, respectively.

[0029] The site-directed mutagenesis primers for introducing the K198E mutation are:

[0030] Forward primer: 5'-CGACGACGCTACCGAAGGTTACTTCCACCA-3' (underlined is the mutated base)

[0031] Reverse primer: 5'-TGGTGGAAGTAACCTTCGGTAGCGTCGTCG-3' (the underline is the mutated base)

[0032] The site-directed mutagenesis primers for introducing the K198Y mutation are:

[0033] Forward primer: 5'-CGACGACGCTACCTATGGTTACTTCCACCA-3' (the underline is the mutated base)

[0034]Reverse primer: 5'-TGGTGGAAGTAACCATAGGTAGCGTCGTCG-3' (the underline is the mutated base)

[0035] The s...

Embodiment 3

[0046] Embodiment 3: Analysis of maltogenic amylase enzyme activity

[0047] (1) Definition of enzyme activity unit

[0048] When using the 3,5-dinitrosalicylic acid method (DNS method) to measure the activity of maltose amylase, the amount of enzyme needed to catalyze the production of 1 μmol of reducing sugar per minute is taken as an activity unit.

[0049] (2) Enzyme Activity Determination Steps

[0050] Preheating: Take 2mL of 0.5% soluble starch solution (50mM pH5.5 citric acid buffer) in a test tube and place it in a 60°C water bath to preheat for 10min.

[0051] Reaction: Add 0.1mL sample enzyme solution, oscillate evenly, accurately time 10min, add 3mL DNS, oscillate evenly, put into ice water to terminate the reaction, boil in a boiling water bath for 7min. cool down.

[0052] Measurement: add distilled water to the above reaction system and adjust the volume to 15mL, and mix well. The absorbance was measured at a wavelength of 540nm and the enzyme activity was c...

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Abstract

The invention discloses preparation of a maltogenic amylase mutant and application thereof and belongs to the field of enzyme engineering. According to the maltogenic amylase mutant constructed in theinvention, maltose content can be increased to 97% or higher while a conversion reaction is carried out for 10 hours in the secondary saccharification process when the addition amount of dry starch is 20U / g, and the content of trisaccharides and tetrasccharides tends to be 0 after reacting for 5 hours. Compared with wild-type maltogenic amylase, the maltogenic amylase mutant has high conversion efficiency and less conversion by-products, and also has important industrial application potential.

Description

technical field [0001] The invention relates to the preparation and application of a maltose amylase mutant, which belongs to the field of enzyme engineering. Background technique [0002] Maltogenic amylase (maltogenic amylase or maltogenase, EC 3.2.1.133) is a member of the GH-H family of glycoside hydrolases. At present, the main bacterial sources of maltogenic amylase are Bacillus stearothermophilus, Bacillus cereus, Bacillus subtilis, Bacillus licheniformis, Thermus vulgaris) and Thermus sp. Maltogenic amylases from different sources also have great differences in properties. The maltogenic amylase currently used in the preparation of malt syrup and anti-aging of bread is mainly derived from Bacillus stearothermophilus. [0003] 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 hygro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12P19/12C12P19/14
CPCC12N9/2402C12P19/12C12P19/14C12Y302/01133
Inventor 黄海军邓希何球山
Owner 湖南金悦降解塑料制品有限公司
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