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Fungal alpha-amylase variant with high maltose generation rate and preparation method for fungal alpha-amylase variant

A high maltose and amylase technology, applied in the field of enzyme engineering, can solve problems such as methods for improving the maltose production rate of fungal alpha-amylase and the like

Active Publication Date: 2016-10-26
合肥停弦渡生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Novozymes' patent "Fungamyl-like α-amylase variant" (patent publication number: CN1654641A) discloses an α-amylase variant used to produce a parental Fungamyl-like α-amylase, the amylase Derived from Aspergillus oryzae, this amylase variant has improved thermostability relative to the parent, but does not involve a method for increasing the maltose production rate of the fungal α-amylase

Method used

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  • Fungal alpha-amylase variant with high maltose generation rate and preparation method for fungal alpha-amylase variant
  • Fungal alpha-amylase variant with high maltose generation rate and preparation method for fungal alpha-amylase variant
  • Fungal alpha-amylase variant with high maltose generation rate and preparation method for fungal alpha-amylase variant

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preparation example Construction

[0087] Preparation of fungal α-amylase variants

[0088] Extract the plasmid DNA in the above-mentioned positive recombinants, use restriction endonucleases EcoRI and NotI to carry out double digestion and connect with the expression plasmid vector pET-28a (+) of the same double digestion, and use the chemical transformation method to The ligation product was introduced into the competent cells of the recipient strain E.coli JM109 to obtain the recombinant plasmid pET-Roamy, which was extracted and introduced into the competent cells of the expression host strain E.coli BL21 to obtain the recombinant plasmid carrying the α-amylase variant coding gene. Recombinant Escherichia coli. The above-mentioned recombinant Escherichia coli is fermented and cultured and induced to express at low temperature to obtain a fermented cell liquid. The cells in the above-mentioned cell liquid were collected by centrifugation and crushed by ultrasonic crushing method. The α-amylase in the cell c...

Embodiment 1

[0091] Cloning of parental fungal α-amylase gene

[0092] Using conventional DNA cloning techniques (Sam Brook J, Fritsch E F. Molecular Cloning Experiment Guide (Second Edition). Jin Dongyan, Li Mengfeng, Hou Yunde, etc. translation. Beijing: Science Press, 1998), extract Rhizopus, Specifically, the total RNA of Rhizopus oryzae F0071 was cloned by reverse transcription technology to obtain the cDNA of the parental α-amylase coding gene, and the cDNA sequence was ligated into the pMD18-simple vector, reacted at 16°C for 4 hours, and then passed the calcium chloride transformation method Introduce E.coli JM109 competent cells, use resistant LB plates to screen positive transformants and obtain recombinant plasmids. The recombinant plasmid was extracted and Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to complete the sequence determination of the target gene. The sequence of the parent Rhizopus oryzae α-amylase coding gene sequence was determined as SEQ ID NO.1.

[...

Embodiment 2

[0095] Construction of variant R333H

[0096] Based on the parent Rhizopus oryzae α-amylase information described in the sequences SEQ ID NO.1 and SEQ ID NO.2, the codon of Y was replaced by the codon of L through primer design, using a commercially available site-directed mutagenesis kit, Mutations were performed according to the instructions provided by the manufacturer (Shanghai Beyond Biotechnology Co., Ltd.) to construct variants of Rhizopus oryzae α-amylase as shown in the sequences SEQ ID NO.1 and SEQ ID NO.2.

[0097] Parent Rhizopus oryzae α-amylase coding gene has been in recombinant plasmid pET-Roamy, with recombinant plasmid pET-Roamy as template, using Pfu DNA polymerase and primer 1 (SEQ ID NO.3) and primer 2 (SEQ ID NO.3) .4) Perform PCR amplification.

[0098] Primer 1: 5ˊ-GTAACGATCCAAACAACCACGAGGTCTTATGGACC-3ˊ

[0099] Primer 2: 5ˊ-GGTCCATAAGACCTCGTGGTTGTTTGGATCGTTAC-3ˊ

[0100] The PCR product was digested with methylase DpnI and then poured into E.coli DH...

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Abstract

The invention discloses a fungal alpha-amylase variant with high maltose generation rate and a preparation method for the fungal alpha-amylase variant. The alpha-amylase variant is obtained by substituting one and / or multiple areas and / or positions of areas 77-81, areas 135-140, areas 214-220 and areas 331-335 correspondingly shown as SEQ ID NO:2, and has alpha-amylase activity. Compared with parental fungal alpha-amylase, the fungal alpha-amylase variant with high maltose generation rate has the advantages that the maltose content of a starch hydrolysate of the fungal alpha-amylase variant is increased by about 5 percent, and the fungal alpha-amylase variant has application advantages in industrial production of high-maltose syrup.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a fungal alpha-amylase variant with high maltose production rate and a preparation method thereof. Background technique [0002] High maltose syrup is a kind of starch sugar mainly composed of maltose (usually >50%), which has a wide range of applications in the food and beverage industry. In the modern starch sugar industry, high maltose syrup is mainly produced by enzymatic conversion of starch. With the improvement of the saccharification process, fungal α-amylase has gradually replaced β-amylase as a saccharification agent for the production of high maltose syrup, and has become a key enzyme preparation in the production process of high maltose syrup. The reason why fungal α-amylase can replace the relatively expensive β-amylase is because of its special product-forming ability-high maltose generating ability. The quality of high maltose syrup depe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/30C12N15/70
CPCC12N9/242C12Y302/01001
Inventor 李松汤斌杨倩田芳源陈阿娜汤文晶葛飞魏胜华陶玉贵
Owner 合肥停弦渡生物科技有限公司
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