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Glucamylase as well as encoding gene and application thereof

A technology that encodes genes and genes, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of insufficient thermal stability, reduced catalytic efficiency, and high cost of glucoamylase, and achieve good industrial application prospects and thermal stability Good results

Inactive Publication Date: 2012-08-08
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the enzyme activity of glucoamylase (AMG) derived from Aspergillus niger is higher, its optimum reaction temperature is far lower than that of thermophilic α-amylase derived from bacteria, thus resulting in higher cost of cooling step in actual production; in addition The thermal stability of glucoamylase (AMG) derived from Aspergillus niger is not good enough, and it is quickly inactivated at high temperature, which reduces the catalytic efficiency of AMG, which leads to an increase in production costs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the acquisition of glucoamylase and its coding gene

[0030] Tengchong thermophilic anaerobic bacteria (strain preservation number AS 1.2430 T =JCM 11007 T ) is a Gram-negative anaerobic eubacterium isolated from hot springs in Tengchong, my country in 2001 by the Institute of Microbiology, Chinese Academy of Sciences. Its optimum growth temperature is 75°C, and its optimum pH is 7-7.5. Together with the Beijing Huada Gene Research Center, the determination and gene annotation of the whole genome sequence of the fungus were completed. The annotation results showed that there were a series of amylolytic enzymes in the genome of the bacteria.

[0031] Extract Tengchong thermophilic anaerobic bacteria (Thermoanaerobacter tengcongensis MB4) (purchased from the Bacteria Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is AS 1.2430 T =JCM 11007 T ) and using it as a template, F: 5'-GAGCCATATGTGTTC...

Embodiment 2

[0033] Embodiment 2, utilize the recombinant bacterium fermentation production glucoamylase that contains glucoamylase coding gene

[0034] 1. Obtaining of recombinant bacteria containing glucoamylase coding gene

[0035] After purification of the glucoamylase encoding gene obtained in Example 1 above, it was double-digested with NdeI and SalI, and the digested product was ligated with the same digested pET42b vector (purchased from Novagen) with T4 ligase overnight at 4°C, and the ligated product was Transform Escherichia coli BL21 (DE3) competent cells to obtain recombinant bacteria. The plasmid sequencing verification of the extracted recombinant bacteria showed that the obtained recombinant bacteria contained the glucoamylase coding gene ttga, indicating that the expression vector was constructed correctly.

[0036] 2. Fermentative production of glucoamylase by recombinant bacteria

[0037] A single colony of the recombinant bacteria obtained in the above step 1 was inoc...

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Abstract

The invention discloses a glucamylase and an encoding gene thereof. The protein is the protein of the following (a) or (b): the (a) is a protein consisting of amino acid sequences as shown in a sequence 2 inside a sequence table; and the (b) is a protein which is obtained by carrying out the substitution and / or the deficiency and / or the addition of one or a few amino acid residues on an amino acid residue sequence of the sequence 2 inside the sequence table, has a glucamylase function and is derived from the (a). Thermoanaerobacter tengcongensis (culture collection number AS 1.2430T=JCM 11007T) is amplified through a genetic engineering technology to obtain the encoding gene of the glucamylase and the coding gene is transferred into escherichia coli BL21(DE3); and in addition, experimental results show that the glucamylase has higher optimum reaction temperature 10 DEG C higher than that of the glucamylase from aspergillus niger, good heat stability and better industrial application prospect.

Description

technical field [0001] The invention relates to a glucoamylase, its encoding gene and application. Background technique [0002] Glucoamylase (1,4-α-D-glucan glucohydrolases, EC 3.2.1.3), also known as glucoamylase (glucoamylase) or γ-amylase (γ-amylase), referred to as glucoamylase. Glucoamylase can hydrolyze α-1,4-glycosidic bonds one by one from the non-reducing end of oligosaccharide or polysaccharide carbon chain to generate β-D-glucose. Glucoamylase is in the last step of starch degradation, and has important application value in food processing, bioethanol production and wine making. [0003] The industrial production of glucose from starch is divided into two consecutive steps of liquefaction and saccharification, in which two main enzymes are used: thermophilic α-amylase from bacteria and glucoamylase (AMG) from Aspergillus niger. In the first step of liquefaction, starch is liquefied into maltodextrin by thermophilic α-amylase at pH 6 and around 100 °C; in the su...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/34C12N15/56C12N15/63C12N5/10C12N1/00C12N1/21C12N15/11C12R1/19
Inventor 马延和郑迎迎薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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