Method of making sucrose phosphorylase(SP) heat-stable

一种蔗糖磷酸化酶、耐热的技术，应用在生物化学设备和方法、植物学设备和方法、酶等方向，能够解决没有获得成功、底物特异性改变、耐热性的提高有限等问题

Active Publication Date: 2006-10-11
EZAKI GLICO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] It has been reported that immobilizing SP enzyme can improve the heat resistance of the enzyme, but the immobilized enzyme has disadvantages, such as the change of substrate specificity, and the improvement of heat resistance by immobilization is limited, so it is hoped to improve the heat resistance of SP enzyme itself. heat resistance
[0018] Theoretical approaches to making common enzymes more thermotolerant, such as the proline theory and amino acid substitutions based on enzyme stereostructure information, have been attempted without certain success

Method used

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  • Method of making sucrose phosphorylase(SP) heat-stable
  • Method of making sucrose phosphorylase(SP) heat-stable
  • Method of making sucrose phosphorylase(SP) heat-stable

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0591] (Example 1: Preparation, screening and sequencing of thermostable sucrose phosphorylase gene)

[0592] Briefly, random mutations were introduced into the sucrose phosphorylase gene derived from Streptococcus mutans, the randomly mutated gene was introduced into Escherichia coli, the randomly mutated sucrose phosphorylase was expressed, and selected from E. Escherichia coli expressing thermostable sucrose phosphorylase capable of synthesizing glucan after heating at 52.5°C for 15 minutes, the gene of thermostable sucrose phosphorylase was isolated from the Escherichia coli, and its sequence was determined.

[0593] Details as follow.

[0594] First, random mutations were introduced into the natural sucrose phosphorylase gene derived from Streptococcus mutans given in SEQ ID NO: 1 by an error-prone PCR method known to those skilled in the art to obtain a randomly mutated SP gene. Under this condition, a sucrose phosphorylase gene usually introduces 1 to 2 random mutation...

Embodiment 2A

[0601] (Example 2A: Preparation of heat-resistant sucrose phosphorylase and determination of heat resistance by site-directed mutagenesis)

[0602] (1) Preparation of thermostable sucrose phosphorylase by site-directed mutagenesis

[0603] Briefly, a library containing the combination of the above 8 mutations was prepared and screened to obtain sucrose phosphorylase with further improved thermotolerance.

[0604] In detail, an expression vector for thermostable sucrose phosphorylase was prepared in the same manner as in Example 1. In this example, a gene encoding a thermostable sucrose phosphorylase substituted at only one position that has been found to contribute to the improvement of thermosistence was prepared, and a gene encoding thermostable sucrose phosphorylase substituted at a combination of 2 to 7 positions was prepared. The gene of the sucrose phosphorylase, and the gene encoding the heat-stable sucrose phosphorylase with all the 8 positions replaced. By way of ex...

Embodiment 2B

[0626] (Example 2B: Preparation of thermostable sucrose phosphorylase with various amino acid residue substitutions and thermostable assay)

[0627] A plasmid containing the modified sucrose phosphorylase gene was prepared in the same manner as in Example 2A, except that the primers used were designed to replace one amino acid in T47, S62, Y77, V128, K140, Q144, N155 and D249 with another amino acid, and each obtained A solution of a modified SP enzyme.

[0628] The heat resistance of these modified SP enzymes was determined by measuring the remaining activity of these modified SP enzymes after heating in the same manner as in Example 2A(2)(i), except that the heating condition was 55° C. for 20 minutes. The results are shown in Table 4B below.

[0629] enzyme solution

residual activity

(%)

T47S

47.3

T47I

50.0

S62P

33.4

S62A

31.0

S62K

26.3

Y77H

22.6

Y77R

34.8

...

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Abstract

It is intended to provide a heat-stable sucrose phosphorylase (SP) obtained by modifying natural SP and a method of preparing this heat-stable SP. Namely, a heat-stable SP which has an amino acid residue differing from the one in natural sucrose phosphorylase at least one position selected from the group consisting of: the positions corresponding respectively to the 14-, 29- and 44-positions in the motif sequence 1; the positions corresponding respectively to the 7-, 19-, 23- and 34-positions in the motif sequence 2; and the position corresponding to the 19-position in the motif sequence 3, and, after heating in a 20 mM Tris buffer solution (pH 7.0) at 55 DEG C for 20 minutes, shows an enzymatic activity amounting to 20% or more of the enzymatic activity of the unheated heat-stable SP at 37 DEG C.

Description

technical field [0001] The present invention relates to a thermostable sucrose phosphorylase and a gene encoding the thermostable sucrose phosphorylase. In addition, the present invention also relates to a method for producing the thermostable sucrose phosphorylase. Background technique [0002] Sucrose phosphorylase (hereinafter also referred to as SP or SP enzyme) is, for example, an enzyme useful in α-glucan synthesis and glucose-1-phosphate (G-1-P) synthesis. [0003] α-glucan (especially insoluble amylose) is considered to have the same function as dietary fiber and can be used in health food. In addition, amylose of linear α-glucan has characteristics such as being able to contain iodine, fatty acid, etc. in its molecule, and thus is expected to be useful in fields such as pharmaceuticals, cosmetics, and hygiene products. Amylose can also be used as a raw material for the production of cyclodextrin and cycloamylose, which have a similar inclusion ability to amylose. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/02C12P19/04
CPCC12N9/1051
Inventor 藤井和俊饭干雅惠柳濑美千代高田洋树鹰羽武史栗木隆
Owner EZAKI GLICO CO LTD
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