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Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase

A metagenomics, animal feces technology, applied in the field of microorganisms and genetic engineering, can solve the problem of less research

Inactive Publication Date: 2014-12-31
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, researchers have successfully screened multiple amylase genes using metagenomic technology, but the research areas are mainly concentrated in water and soil (Delavat et al. Sci Rep.2012,2:354; Liu et al. Mar Biotechnol (NY ).2012,14(3):253-260; Sharma et al. Appl Microbiol Biotechnol. 2010, 86(6):1821-1828), there are fewer studies on animal gastrointestinal microbes (Tasse et al . Genome Res. 2010,20:1605-1612; Ferrer et al. Biotechnol J. 2007,2:207-213)

Method used

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  • Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase
  • Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase
  • Alpha-amylase derived from animal feces metagenome and gene of the alpha-amylase

Examples

Experimental program
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Embodiment 1

[0029] Example 1: Obtaining α-amylase gene

[0030] 1. Construction of a metagenomic library of fecal microbes of Japanese monkeys

[0031] Extract microbial genomic DNA from the feces of Japanese monkeys (refer to the patent "A method for extracting high-molecular-weight genomes from animal feces", publication number 102586234A). After fragmentation, separate by pulse field electrophoresis and agarose gel electrophoresis Recover a DNA fragment with a size of about 40kb, connect the recovered DNA fragment to the fosmid vector pCC1FOS and transfect the host bacteria E.coli EPI300 was spread on an LB plate containing 12.5 μg / mL chloramphenicol, incubated overnight at 37°C to obtain transformants, and the clone library was stored in a 96-well plate and stored at -80°C.

[0032] 2. Functional screening of positive clones producing amylase

[0033] Dilute the cloned bacteria stored in the 96-well plate appropriately, spread it on the TB-chloramphenicol (12.5μg / mL) screening plate contain...

Embodiment 2

[0041] Example 2: Preparation of recombinant α-amylase

[0042] The alpha-amylase gene pl A was connected with plasmid pEASY-E1 to obtain a recombinant expression vector, and then transformed into E. coli BL21 (DE3) to obtain a recombinant strain. Take the recombinant expression vector E. coli The BL21(DE3) strain was inoculated into LB (containing 100μg / mL Amp) culture medium at a 0.1% inoculum, and shaken rapidly at 37°C for 16h. Then the activated bacterial solution was inoculated into fresh LB (containing 100μg / mL Amp) culture solution at 1% inoculum amount, and cultured with rapid shaking for about 2–3h (OD 600 After reaching 0.6-1.0), add a final concentration of 0.5% α-lactose to induce, continue shaking culture at 20°C for about 20 hours or 26°C shaking culture for about 8 hours. Centrifuge at 12000 rpm for 5 min to collect the bacteria. After suspending the bacteria in an appropriate amount of pH7.0 Tris-HCl buffer, ultrasonically disrupt the bacteria in a low-tempera...

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Abstract

The invention relates to alpha-amylase derived from animal feces metagenome and a gene of the alpha-amylase. The alpha-amylase gene is 1458 bp in length and the sequence of the alpha-amylase gene is shown as SEQIDNO.1. The gene encodes 485 amino acids and the sequence of the amino acids is shown as SEQIDNO.2. The alpha-amylase provided by the invention has characters that the optimal operative pH is 5.6; the enzyme activity remains larger than 65% when the pH is ranging from 4.6 to 6.6; the enzyme activity remains larger than 70% after the alpha-amylase is treated at a pH ranging from 4.0 to 8.0 for 1 h; the optimal operative temperature is 50 DEG C; and the alpha-amylase is relatively stable at 37 DEG C, and the enzyme activity remains larger than 80% after the enzyme is kept at 37 DEG C for 50 min. As a novel enzyme preparation, the alpha-amylase provided by the invention can be widely used in forage processing industry, starch processing industry, and the like.

Description

technical field [0001] The invention belongs to the technical field of microbes and genetic engineering, and in particular relates to an alpha-amylase from animal feces metagenome and its gene. Background technique [0002] α-amylase (EC 3.2.1.1) is an endoglucosidase that can hydrolyze α-1,4-glucosidic bonds in starch to produce short-chain dextrins of different lengths and a small amount of low-molecular-weight sugars , so that the viscosity of the starch paste decreases rapidly, that is, it plays the role of reducing the consistency and "liquefying", so it is also called liquefying enzyme. α-amylases are widely found in various organisms such as humans, animals, plants, fungi, and bacteria. The isolation of α-amylases was first reported in 1956. So far, researchers from various countries have isolated and identified more than 120 kinds of α-amylases, among which Mainly derived from microorganisms of the genus Bacillus ( Bacillus ), Aspergillus ( Aspergillus ), Strepto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/26C12N15/70C12N1/21C12R1/19
Inventor 许波黄遵锡杨富亚李俊俊唐湘华周峻沛
Owner YUNNAN NORMAL UNIV