A kind of α-amylase, encoding gene, carrier, host and application thereof

A technology encoding gene and amylase, applied in the fields of bioengineering and enzyme engineering, can solve problems such as the inability to faithfully reflect the function of functional enzyme genes, and the inability to effectively mine enzyme genes by culture methods.

Active Publication Date: 2021-05-07
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the culture method cannot effectively mine the enzyme genes in non-culturable microorganisms, and the metagenomic method cannot faithfully reflect the role of functional enzyme genes in the environment

Method used

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  • A kind of α-amylase, encoding gene, carrier, host and application thereof
  • A kind of α-amylase, encoding gene, carrier, host and application thereof
  • A kind of α-amylase, encoding gene, carrier, host and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Acquisition of α-amylase encoding gene

[0030]Samples were taken at the high temperature stage (62°C) of Luzhou-flavor Daqu, and total RNA was extracted. The specific method is as follows: grind 1 g of Luzhou-flavor Daqu sample into a fine powder in a pre-cooled mortar. The sample was mixed with 4 mL of borate buffer (200 mM sodium borate, 30 mM ethylene glycol tetraacetic acid (EGTA), 1% (w / v) sodium dodecyl sulfate (SDS), 4% (w / v) v) Polyvinylpyrrolidone (PVP), 0.5% (v / v) Nonidet-40 (NP-40), 10 mM β-mercaptoethanol and 0.03% (v / v) RNase inhibitor) and 280 μL proteinase K (20 mg / mL) Mix and keep at room temperature for 2 minutes. RNA from the resulting crude lysate was then centrifuged, precipitated with 70% ethanol, and washed according to the instructions of the RNeasy Midi Kit (Qiagen, Valencia, CA). In addition, the RNA mixture was treated with DNase I (Fermentas, USA) according to the instructions. Use 3 μL of total RNA (approximately 1 μg) as a te...

Embodiment 2

[0031] Example 2: α-amylase bioinformatics analysis

[0032] The protein expressed by the NFAmy13B gene obtained in Example 1 is named α-amylase NFAmy13B, which contains 534 amino acids, its amino acid sequence is shown in SEQ ID NO.2, and the predicted protein size is 61.2KD. The nucleotide sequence of NFAmy13B gene was predicted according to SignalP-5.0Server (http: / / www.cbs.dtu.dk / services / SignalP / ), which showed that it had no signal peptide. According to the gene bank sequence comparison, the enzyme with the highest amino acid sequence similarity to the α-amylase NFAmy13B is a putative α-amylase derived from Byssochlamys spectabilis (GenBank number: XP_028489551.1), with a similarity of 77.6%. Amylase was derived from the analysis of genome information, and the enzymatic properties were not analyzed. The second is the intracellular fungal amylase AmyD (GenBank number: XP_001389762.2) derived from Aspergillus niger, with a similarity of 64.4%. Phylogenetic tree analysis ...

Embodiment 3

[0033] Example 3: Expression of α-amylase gene

[0034] NFAmy13B was amplified by forward primer NFABf (5'-CACCATGAAGTCCCTCCTCTGCTG-3') and reverse primer NFABr (5'-CTAGTGCTTGTAGATATCCGAGTC-3') using the cDNA containing open reading frame 22984 in the Luzhou-flavor Daqu cDNA library as a template Gene. The specific PCR reaction system is: 47 μL of Mix (green) (TsingKe, Beijing), 1 μL of 10 μM forward and reverse primers, and 1 μL of DNA template. PCR amplification conditions were: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 50°C for 15 s, extension at 72°C for 25 s, skip to step 2 and cycle 30 times, and keep at 72°C for 5 min. The amplified product detected by agarose gel electrophoresis was about 1.6kb, which was consistent with the expected NFAmy13B gene containing 1605 bases. 50-100 ng of the amplified product was ligated with pDE2 plasmid (TsingKe, Beijing) at 22-30° C. for 1-5 min to construct a recombinant expression vector.

[003...

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Abstract

The present invention provides an α-amylase encoding gene, α-amylase, a recombinant vector comprising the α-amylase encoding gene, a host comprising the α-amylase encoding gene or the recombinant vector, and preparation The method of the α-amylase, and confirmed that the α-amylase has hydrolysis activity to various starch substrates, so the α-amylase and the host can be used in hydrolyzing starch, and can be specifically applied to the washing industry , textile industry and food industry, etc., providing a new choice for starch hydrolysis that can be applied to washing, textile and food industries.

Description

technical field [0001] The invention belongs to the field of bioengineering and enzyme engineering, and relates to alpha-amylase, coding gene, carrier, host and application thereof. Background technique [0002] α-Amylase (E.C.3.2.1.1.) is the most important enzyme among industrially applied enzymes, accounting for 25% of all industrial enzymes. α-amylase is an endoamylase that acts on the α-1,4-glucosidic bond of starch substrate to release oligosaccharides such as glucose, maltose and maltotriose. As a representative of the glycoside hydrolase 13 family (GH13), it can be divided into GH13_1, 5, 6, 7, 15, 24, 27, 28, 32, 36, and 37 subfamilies according to sequence similarity and catalytic function. Among them, fungal amylases belonged to GH13_1 and 5 subfamilies. GH13_1 only contains α-amylases from fungi and yeast, and most α-amylases are extracellular enzymes; the GH13_5 subfamily includes liquefied amylases from bacteria, fungal intracellular enzymes and some archaeal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/70C12N1/21
CPCC12N9/2417C12N15/70C12Y302/01001
Inventor 赵海易卓林陈兰钗方扬靳艳玲何开泽
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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