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Glucose isomerase mutant

A glucose isomerase and mutant technology, applied in the field of glucose isomerase mutants, can solve the problems of low activity, unused industrial production, and unsatisfactory heat resistance of high-temperature resistant glucose isomerase, and achieve high catalytic performance active effect

Active Publication Date: 2009-04-01
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The thermostable glucose isomerase of these or other sources is all unsatisfactory, and activity is low, has not been seen to be used in industrialized production yet

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Amplification of parental genes and construction of pGEMT-TS

[0028] Primers T1 and T2 were designed according to the gene sequence of the gene bank (GenBank L09699) (see Table 1). The glucose isomerase parent gene was amplified from T. saccharolyticum ATCC49915 (purchased from ATCC, USA) using primer pair T1 and T2.

[0029] Amplification conditions are: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% TritonX-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1, 400 nM primer T2, 1.5U Pfu DNA polymerase (Promega, USA), pick a little T. saccharolyticum thallus with an inoculation loop , and then adjust the reaction volume to 50 μl with sterile water.

[0030] The PCR amplification reaction program was: 95°C for 3 minutes, 40 cycles: 95°C for 50 seconds, 50°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified product (about 1.5 kb in length) was connected to the vector pGEMT-Easy t...

Embodiment 2

[0031] Example 2: Site-directed mutation of glucose isomerase position 139

[0032] For the technique of site-directed mutagenesis, refer to the descriptions of Ho et al. (Gene 77:51-59, 1989 and White et al. (PCR Protocol: current methods and applications. Totowa, N.J.: Humana Press, 1993).

[0033] Using the plasmid pGEMT-TS (see Example 1) as a template, design the primer pair 139FF and 139FR (see Table 1), and mutate the Trp (W) at position 139 in the parental amino acid sequence to Phe (F) to obtain the mutant MGI-W139F. See Example 1 for primer pair T1 and T2.

[0034] The T1FR fragment was amplified by primer pair T1 and 139FR; the FFT2 fragment was amplified by primer pair 139FF and T2. The amplification reaction conditions are: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 139FR or 400 nM primer 139FF and 400 nM primer T2, 1.5 U Pfu DNA pol...

Embodiment 3

[0036] Example 3: Site-directed mutation of glucose isomerase position 182

[0037] For the technique of site-directed mutagenesis, refer to the descriptions of Ho et al. (Gene 77:51-59, 1989) and White et al. (PCR Protocol: current methods and applications. Totowa, N.J.: Humana Press, 1993).

[0038] Using the plasmid pGEMT-TS (see Example 1) as a template, design the primer pair 182AF and 182AR (see Table 1), and mutate the Arg(R) at position 182 in the parental amino acid sequence to Ala(A) to obtain the mutant MGI-R182A. See Example 1 for primers T1 and T2.

[0039] Using the primer pair T1 and 182AR, amplify the T1AR fragment; using the primer pair 182AF and T2, amplify the AFT2 fragment. The amplification reaction conditions are: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 400nM primer T1 and 400nM primer 182AR or 400nM primer 182AF and 400nM primer T2, 1.5U PfuDNA polymerase, 20...

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Abstract

The invention discloses a series of Thermoanaerobacterium saccharolyticum with heat resistance and high catalytic activity improved by utilizing a gene mutation technology. The Thermoanaerobacterium saccharolyticum can be used for directly generating high fructose syrup with fructose content equal to or more than 55 weight percent, or used for generating high fructose syrup with fructose content lower than 55 weight percent.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of June 16, 2004, an application number of 200410047865.X, and an invention title of "Glucose Isomerase Mutant". Technical field: [0002] The present invention relates to the fields of molecular biology and biotechnology, in particular to a method for preparing high-activity or high-activity and heat-resistant glucose isomerase mutants using gene mutation technology, the obtained mutants and applications thereof. Background technique: [0003] Glucose isomerase (Glucose isomerase, E.C.5.3.1.5, referred to as GI), or xylose isomerase (Xyloseisomerase) is a key enzyme in the pentose sugar fermentation pathway. This enzyme is one of the most important enzyme preparations (Kaneko, et al., Biosci Biotechnol Biochem, 64: 940-947, 2000). The food industry uses this enzyme to prepare fructose syrup. [0004] Enzymatic production of fructose syrup, the content of fructose in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/92C12N15/61C12P19/24
Inventor 王骏傅荣昭沈冬金彩科刘兆明陈军明
Owner BIORIGHT WORLDWIDE
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