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Acid resistant fungi alpha-amylase TaAMY, gene and applications thereof

An acid-resistant and amylase technology, applied in the field of genetic engineering, can solve the problems of large gap, lack of production and market competitiveness, and low production level of fungal α-amylase

Active Publication Date: 2014-01-15
JIANGSU YINONG BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the production level of fungal α-amylase in domestic enzyme preparation companies is relatively low. Compared with several foreign enzyme preparation companies that are at the world's leading level (the fermentation level can reach about 2000-3000U / g), there is a big gap, and there is no production and market competitive advantage

Method used

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  • Acid resistant fungi alpha-amylase TaAMY, gene and applications thereof
  • Acid resistant fungi alpha-amylase TaAMY, gene and applications thereof
  • Acid resistant fungi alpha-amylase TaAMY, gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0094] Embodiment 1, the mutagenesis and cultivation of fungal alpha-amylase Aspergillus niger strain

[0095] Aspergillus niger strain AN0921 (strain preservation number ACCC30132), which is native to fungal α-amylase, was cultured on a PDA plate, and then used plasma (ARTP), ultraviolet light, and diethyl sulfate (DES) for individual and compound mutagenesis After treatment, a fungal α-amylase mutant strain TaAN0921 was screened.

[0096] The mutant strain is fermented and cultivated, and its fermentation medium composition is as follows:

[0097] Peptone 2%, yeast powder 0.5%, dextrin 10%, sodium glutamate 1.5%, potassium diammonium phosphate 0.15%, magnesium sulfate 0.10%

[0098] The above fermentation culture is based on sterilization at 121° C. and 0.1 MPa for 30 minutes, inoculation after cooling, and cultivation at 30° C. and 200 rpm for 4 to 5 days.

[0099] After the fermentation, take an appropriate amount of enzyme liquid for testing. The test results show that th...

Embodiment 2

[0101] Example 2, Cloning of Aspergillus niger (TaAN0921) α-amylase gene

[0102] According to reverse transcriptase SuperScript TM The operating instructions of III Reverse Transcriptase, use the RNA extraction kit to extract the total RNA of Aspergillus niger (TaAN0921) and synthesize the first-strand cDNA. Using cDNA as a template, primers were designed for PCR amplification, and the PCR product was digested with EcoRI and NotI, and then connected with the Pichia pastoris expression vector pPICzαA that had undergone the same digestion, and the ligated product was transformed into Escherichia coli Topl0 competent cells, After screening by the antibiotic Zeocin, positive clones were obtained. Plasmids of positive clones were extracted and sequenced. Sequencing results showed that the cloned DNA insert contained the complete open reading frame of the fungal α-amylase gene. The total length of the fungal α-amylase gene (NTaAMY) is 1704bp, encoding 567 amino acids, and the re...

Embodiment 3

[0108] Embodiment 3, Pichia pastoris engineered bacteria construction comprising novel acid-resistant fungal α-amylase gene NTaAMY

[0109] The above-mentioned recombinant expression vector (NTaAMY-pPICzαA) was linearized with SacI, and the linearized recombinant vector was electroporated to transform Pichia pastoris X33. After electroporation, it was spread on a YPD plate containing Zeocin for screening to obtain a recombinant strain of Pichia pastoris NTaAMY-pPICzαA-X33.

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Abstract

The invention relates to the fields of gene engineering and fermentation engineering, and specifically relates to an acid resistant fungi alpha-amylase TaAMY, gene and applications thereof. The invention provides a novel acid resistant fungi alpha-amylase TaAMY, which has an amino acid sequence represented by the SEQ ID No.1, also provides a gene NTaAMY, which encodes the novel acid resistant fungi alpha-amylase mentioned above and has a nucleotide sequence represented by the SEQ ID No.2, and also provides a recombinant vector containing the gene mentioned above, a recombinant bacterium strain and applications thereof. The novel acid resistant fungi alpha-amylase can reach a relatively high fermentation level after the high-level expression, and can be widely applied to fields of starch sugar making, brewing, baking, medicine, and feed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an acid-resistant fungal alpha-amylase TaAMY, its gene and application. Background technique [0002] Fungal α-amylase is produced by fungi such as Aspergillus niger, Aspergillus oryzae, Rhizopus, etc. It can randomly hydrolyze the α-1,4 glycosidic bonds in starch, and also hydrolyze maltotriose. The final product is mainly maltose and some oligomerization Oligosaccharides and a small amount of glucose. Generally, the thermal stability of fungal α-amylase is not as good as that of bacterial α-amylase, and has the characteristics of mild reaction temperature and narrow pH range. [0003] Since the 1950s, the United Kingdom and the United States have successively applied fungal α-amylase as a food additive to the bread production industry. Due to the characteristics of fungal α-amylase and the rapid development of modern food industry and fermentation industry, fungal α-amylase...

Claims

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Application Information

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IPC IPC(8): C12N9/30C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/242C12Y302/01001
Inventor 张慧君王俊峰王菲菲
Owner JIANGSU YINONG BIOLOGY CO LTD
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