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Raw maltogenic amylase production bacterial strain

A technology of maltose amylase and bacteria, which is applied in the field of genetic engineering, can solve problems such as the reduction of the final yield of maltose and the reduction of product purity, and achieve the effects of improved temperature stability, high catalytic efficiency and low by-products

Active Publication Date: 2019-02-12
湖南金代科技发展有限公司
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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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  • Raw maltogenic amylase production bacterial strain
  • Raw maltogenic amylase production bacterial strain
  • Raw maltogenic amylase production bacterial strain

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Preparation of wild raw maltogenic amylase.

[0022] (1) Construction of Maltogenic Amylase Recombinant Bacteria

[0023] 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 e...

Embodiment 2

[0027] Embodiment 2: Preparation of raw maltogenic amylase mutants

[0028] (1) Replace tryptophan (Trp) at position 210 in maltogenic amylase with phenylalanine (Phe), denoted as W210F.

[0029] The site-directed mutagenesis primers for introducing the W210F mutation are:

[0030] Forward primer: 5'-TGACATCTCTAAC TTC GACGACCGTTACGA-3' (the underline is the mutated base)

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

[0032] The pET24a-amyM plasmid was used as a template for PCR reaction. All reactions were carried out in a 50 μL system, and the reaction conditions were: pre-denaturation at 94°C for 4 min; followed by 30 cycles (94°C for 10 s, 50°C for 10 s, 72°C for 7 min and 20 s); extension at 72°C for 10 min; and finally incubation at 4°C. The PCR products were digested with DpnⅠ (Fermentas Company), and transformed into Escherichia coli JM109 competent cells, respectively, and the transformed products were sprea...

Embodiment 3

[0040] Embodiment 3: Enzyme activity analysis of raw maltose amylase

[0041] (1) Definition of enzyme activity unit

[0042] When using the 3,5-dinitrosalicylic acid method (DNS method) to measure the activity of maltogenic amylase, the amount of enzyme needed to catalyze the production of 1 μmol of reducing sugar per minute is taken as an activity unit.

[0043] (2) Enzyme Activity Determination Steps

[0044] 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.

[0045] 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.

[0046] 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 w...

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Abstract

The invention discloses a raw maltogenic amylase production bacterial strain and belongs to the technical field of genetic engineering. According to the raw maltogenic amylase production bacterial strain disclosed by the invention, raw maltogenic amylase for preparing maltose is constructed at first, the maltose content of the maltogenic amylase can be improved to 96% or above when a conversion reaction is performed for 10 h, the contents of trisaccharide and tetrasccharide is close to 0 when the reaction is performed for 5 h, and the characteristics of low byproducts, high catalytic efficiency and the like can be achieved. By means of self-assembly oligopeptide modification, mutant temperature stability is remarkably improved, a half-life period is prolonged to 304 h from 97 h, and the optimum temperature is more suitable for a temperature required by a saccharifying process. The raw maltogenic amylase production bacterial strain disclosed by the invention successfully realizes heterogeneous expression of the maltogenic amylase, is favorable for the large-scale preparation of the raw maltogenic amylase suitable for producing maltose.

Description

technical field [0001] The invention relates to a strain producing maltogenic amylase, which belongs to the technical field of genetic 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, thermophilic actinomycetes ( Thermus vulgaris) and Thermus sp. There are also great differences in the properties of maltogenic amylases from different sources. 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 ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/62C12N15/56C12N15/70C12N9/24C12P19/14C12P19/12C12R1/19
CPCC07K2319/00C12N9/2402C12N15/70C12P19/12C12P19/14C12Y302/01133
Inventor 谭启程张国军钟红霞
Owner 湖南金代科技发展有限公司
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