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A kind of amylase tlamy5 derived from fungus and its gene and application

An amylase and gene technology, applied in the field of genetic engineering, can solve the problems of high price of pullulan polysaccharide and increased production cost, etc.

Active Publication Date: 2020-10-16
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Maltotriose has the characteristics of high water absorption, acid resistance and heat resistance, and is favored by more and more food and beverage production. Although pullulan can be used as a substrate to obtain high-purity maltotriose, the price of pullulan Expensive, increases production costs

Method used

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  • A kind of amylase tlamy5 derived from fungus and its gene and application
  • A kind of amylase tlamy5 derived from fungus and its gene and application
  • A kind of amylase tlamy5 derived from fungus and its gene and application

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Experimental program
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Embodiment 1

[0050] Cloning of embodiment 1 amylase coding gene T1Amy5

[0051] Extraction of Genomic DNA from Talaromyces leycettanus JCM12802

[0052]Cloning primers were designed, and PCR amplification was performed using the total DNA of Talaromyces leycettanus JCM12802 as a template. The PCR reaction parameters are: 95°C for 5min; 35 cycles of 94°C for 30sec, 60°C for 30sec, 72°C for 2min, and 72°C for 10min. A fragment of about 1800bp was obtained, which was recovered and sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0053] Table 1 Primers required for gene cloning in this experiment

[0054]

Embodiment 2

[0055] The acquisition of embodiment 2 amylase cDNA

[0056] Extraction of Talaromyces leycettanus JCM1280, total RNA, using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then design primers 12802GH13-5-F and 12802GH13-5-R (see Table 1) for amplifying the open reading frame, amplify the single-stranded cDNA, and obtain the cDNA of amylase Sequence, the amplified product was recovered and sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0057] After comparing the genome sequence and cDNA sequence of amylase, it is found that the gene contains 8 introns, the cDNA is 1833bp long, encodes 610 amino acids and a stop codon, and the N-terminal 20 amino acids are its signal peptide sequence. The gene encoding amylase isolated and cloned from Talaromyces leycettanus JCM1280 is a new gene.

Embodiment 3

[0058] The construction of embodiment 3 amylase engineering strains

[0059] (1) Construction of expression vector and expression in yeast

[0060] Taking the cDNA of amylase T1Amy5 with the correct sequence as template, the primers 12802GH13-5-F and 12802GH13-5-R (see Table 1) with EcoR I and Not I restriction sites were designed and synthesized. The coding region of the mature protein is amplified. And use EcoR I and Not I to cut the PCR product, connect into the expression vector pPIC9 (Invitrogen, San Diego), the sequence of the amylase T1Amy5 mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and form a correct reading with the signal peptide The framework was constructed into a yeast expression vector pPIC9-TlAmy5, which was transformed into Escherichia coli competent cells Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-sc...

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Abstract

The invention relates to the field of gene engineering, in particular to amylase TlAmy5 from fungi as well as a gene and application thereof. The amino acid sequence of the amylase TlAmy5 is shown as SEQ ID NO.1 or SEQ ID NO.2. The invention provides a novel amylase gene. The amylase coded by the novel amylase gene has good properties, and can be applied to industries such as feed, food and medicine. The production of the good-property amylase suitable for industrial application can be realized by utilizing the gene engineering measure according to the technical scheme.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a fungal amylase T1Amy5 and its gene and application. Background technique [0002] Amylase is a biocatalyst with a wide range of uses, which can be used in bread making industry, starch saccharification and liquefaction, textile desizing, paper making, cleaning agent industry, chemistry, clinical medicine analysis and pharmaceutical industry, etc. The amylase family includes alpha-amylases, beta-amylases and glucoamylases. α-amylase is an endonuclease, which cuts the α-1,4 glycosidic bonds inside the starch molecule in an irregular manner, and makes the starch generate dextrin and oligosaccharides. It is a calcium ion-dependent enzyme. β-amylase sequentially cuts maltose from the non-reducing end, which is an exonuclease. Glucoamylase is an exonuclease acting on α-1,4 glycosidic bonds, cleaving glucose molecules from non-reducing sugar ends. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/30C12N15/56C12N15/81C12N1/19
CPCC12N9/242C12Y302/01001
Inventor 姚斌罗会颖张多多王亚茹王苑黄火清柏映国苏小运涂涛
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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