Pyrococcus produced high temperature alpha-glucosidase and enzyme producing method thereof

A technology of glucosidase and high temperature, applied in the field of microbial engineering, can solve the problems of few research reports on the properties and applications of α-glucosidase

Inactive Publication Date: 2010-12-15
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, domestic research mainly focuses on pharmaceutical α-glucosidase inhibitors, but there are few research reports on the properties and applications of α-glucosidase, only a few reports such as Thermus thermophilus and Aspergillus niger

Method used

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  • Pyrococcus produced high temperature alpha-glucosidase and enzyme producing method thereof
  • Pyrococcus produced high temperature alpha-glucosidase and enzyme producing method thereof
  • Pyrococcus produced high temperature alpha-glucosidase and enzyme producing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1. A kind of thermococcus (Thermococcus sp.HJ21) produces the method for high-temperature α-glucosidase, and its steps are as follows:

[0060] (1) The seed solution is inoculated on the enzyme-producing medium with an inoculation amount of 2.5%, and cultured at 85° C. for 21 hours. The culture solution is filtered with filter paper to remove sulfur particles, and centrifuged at 10,000 r / min for 10 minutes to remove the bacteria. supernatant;

[0061] (2) Add ammonium sulfate to the supernatant to 65% saturation, overnight at 4°C; centrifuge at 10000r / min for 30min, discard the supernatant, dissolve the precipitate with 25mM pH7.5 Tris-HCl solution, dialyze overnight at 4°C, Centrifuge at 12000r / min for 10min, and the obtained supernatant is high-temperature α-glucosidase.

Embodiment 2

[0062] Example 2. A high-temperature α-glucosidase produced by the method described in Example 1, the enzymatic properties of the high-temperature α-glucosidase are: the optimum temperature for the enzyme action is 100°C, and there is still 60% enzyme activity; keep 90% of enzyme activity at 80°C for 4 hours, keep 80% of enzyme activity at 90°C for 1 hour; Ca 2+ It can reduce the thermal stability of high-temperature α-glucosidase; the enzyme is suitable for pH 7.0, and the enzyme activity is more than 70% at pH 6.0-8.0; the enzyme is stable at pH 5.0-8.0; Fe 3+ 、K + 、Ag + , DTT and EDTA can activate the enzyme, Cu 2+ 、Al 3+ 、Ni 2+, iodoacetic acid and SDS can inhibit the enzyme; 1mM Hg 2+ Completely inhibits the activity of the enzyme.

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Abstract

The invention discloses a method for high temperature Alpha-glucosidase generated through hot coccus (Thermococcus sp.HJ21). The invention also discloses a high temperature Alpha-glucosidase product which is obtained through the method, and the optimum temperature for the high temperature Alpha-glucosidase to act is 100 DEG C; the high temperature Alpha-glucosidase is warmed for 4 hours at 80 DEGC and retained 90 percent of the high temperature Alpha-glucosidase vitality, and the high temperature Alpha-glucosidase is warmed for an hour at 90 DEG C and retained 80 percent of the high temperature Alpha-glucosidase vitality; the suitable action PH of the high temperature Alpha-glucosidase is 7.0; the high temperature Alpha-glucosidase is stable in the PH value of 5.0 to 8.0; Fe<3+>, K<+>, Ag<+>, DTT and EDTA have the activation to the high temperature Alpha-glucosidase, and Cu<2+>, Al<3+>, Ni<2+>, iodoacetic acid and SDS have the inhibitory action to the high temperature Alpha-glucosidase; and the high temperature Alpha-glucosidase vitality is absolutely inhibited through 1mM Hg<2+>. The high temperature Alpha-glucosidase obtained through the invention is mainly applied in the production of the oligoisomaltose in the industry, and used as the diastatic enzyme preparation which can produce the high glucose syrup with the Alpha-amylase together in the starch processing industry.

Description

technical field [0001] The invention relates to a strain enzyme production method and a product thereof, in particular to a thermococcus (Thermococcussp. Background technique [0002] α-glucosidase (α-glucosidase EC 3.2.1.20), also known as α-transglucosidase (EC 2.4.1.24), can cleave α-1 from the non-reducing end of oligosaccharide substrates , 4 glycosidic bonds, release glucose, or transfer the free glucose residues to another carbohydrate substrate with α-1,6 glycosidic bonds, so as to obtain non-fermentable isomaltooligosaccharides (IMO), sugar lipids or glycopeptides. The enzyme has both hydrolysis ability and transfer ability. The characteristics of α-glucosidase glycosidic bond hydrolysis and transfer activity may be caused by the difference of enzyme binding and catalytic site structure. α-glucosidase is widely distributed in nature, with various types and different properties, and exists in almost all organisms. It has important physiological functions in the de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C12N1/20C12R1/01
Inventor 王淑军李华钟杨磊吕明生陈丽刘姝房耀维
Owner HUAIHAI INST OF TECH
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