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Method for producing high-temperature alpha-amylase by thermococcaceae aeropyrum pernix and product thereof

A technology of thermophilic archaea and cocci, applied in the direction of biochemical equipment and methods, enzymes, bacteria, etc.

Inactive Publication Date: 2009-11-04
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few domestic studies on the physiology of marine hyperthermophilic microorganisms and high-temperature amylase enzymology, and there are no reports on the production of α-amylase by Thermococcus siculi at home and abroad.

Method used

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  • Method for producing high-temperature alpha-amylase by thermococcaceae aeropyrum pernix and product thereof
  • Method for producing high-temperature alpha-amylase by thermococcaceae aeropyrum pernix and product thereof
  • Method for producing high-temperature alpha-amylase by thermococcaceae aeropyrum pernix and product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. A Thermococcus siculi HJ21 (Thermococcus siculi HJ21) CCTCC NO: M 207010. The strain has the following characteristics: the strain is a strictly anaerobic coccus with a diameter of 0.5-1um; the growth temperature range of the strain is 55-94°C, and its optimum growth temperature is 88°C; the optimum growth pH range is 6.5-7.0, low The strain does not grow at 4.5 or higher than 9.0; the growth NaCl concentration range is 1-5%, 2% is the most suitable NaCl concentration, no NaCl and higher than 5% do not grow; the strain grows well in protein-rich substrates; starch, Maltose, glucose, sucrose, cellobiose, lactose, glycogen, gelatin promote the growth of the strain.

Embodiment 2

[0064] Example 2. Utilize the method for thermostable acid α-amylase produced by Thermococcus thermophilic archaea HJ21 CCTCC NO: M207010 described in Example 1, the steps are as follows,

[0065] (1) Inoculate the strain in YPS medium with a pH of 6.5, and cultivate it at 88°C for 8-9 hours; take out the fermentation broth and cool it to room temperature, filter and remove sulfur particles with a vacuum circulation pump, and then centrifuge at 11000rpm / min for 20min. supernatant to obtain crude enzyme liquid;

[0066](2) Add ammonium sulfate powder to the crude enzyme solution to 70% saturation, stir while adding, continue to stir for 15 minutes after the addition, and then place it in a 4°C incubator for 12 hours; then 11000r / min, 20min Collect the precipitate by centrifugation, add an appropriate amount of 50mM Tris-HCl buffer solution with a pH of 7.5 to dissolve, put the salted out solution in a dialysis bag, place it at 4°C, and dialyze it with a 50mM Tris-HCl buffer so...

Embodiment 3

[0067] Example 3. The high temperature-resistant acid alpha-amylase produced by the method described in embodiment 2, described high temperature resistant acid alpha-amylase has the following characteristics:

[0068] (1) The optimum action temperature of the enzyme is 95°C, and the enzyme has good thermal stability. The enzyme keeps the enzyme activity unchanged at 90°C for 2 hours, 75% of the enzyme activity at 100°C for 1 hour, and 110°C for 2 hours. 40% enzyme activity, is a high temperature resistant α-amylase;

[0069] (2) The optimal pH of the enzyme is 5.0, and the most stable pH of the enzyme is 5.5, which is an acid α-amylase;

[0070] (3)K + 、Sr 2+ , Mg 2+ 、Na + It has a certain promoting effect on the activity of α-amylase; Cu 2+ , Pb 2 + , Hg 2+ , Zn 2+ Strong inhibitory effect on α-amylase;

[0071] (4) IAA, PMSF, SDS have a strong inhibitory effect on the enzyme, EDTA-Na and 2-hydroxyethanol have an inhibitory effect on the enzyme activity.

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Abstract

The invention relates to a Thermococcus siculi HJ21 (Thermococcus siculi HJ21) CCTCC NO: M 207010. The strain is a strictly anaerobic coccus with a diameter of 0.5-1μm; the growth temperature range of the strain is 55-94°C, and the optimum growth temperature is 88°C; the optimum growth pH range is 6.5-7.0, lower than 4.5 or higher than 9.0 The strain does not grow; the growth NaCl concentration range is 1-5%, 2% is the most suitable NaCl concentration, no NaCl and higher than 5% do not grow; the strain grows well in a protein-rich substrate; starch, maltose, glucose, sucrose, Cellobiose, lactose, glycogen, gelatin promote strain growth. The invention also discloses a method for producing high-temperature-resistant acid alpha-amylase by using Thermococcus thermophilic archaea HJ21CCTCC NO: M207010 and the produced high-temperature-resistant acid alpha-amylase.

Description

technical field [0001] The present invention relates to a thermophilic archaea of ​​the genus Thermococcus HJ21 isolated from deep-sea hydrothermal vents (preserved in China Center for Type Culture Collection CCTCC on January 29, 2007, and the preservation number is CCTCC NO: M 207010); the present invention The invention also relates to a method and a product for producing a high-temperature resistant α-amylase by the strain. Background technique [0002] α-amylase, systematically named α-1,4-glucan-4-glucanohydrolase (α-1,4-glucan-4-glucanohydrolase EC.3.2.1.1), is the most important industrial enzyme One of the preparations, accounting for about 25% of the entire enzyme preparation market, has been widely used in many industries such as food, fermentation, textile, papermaking and pharmaceuticals; but most of the α-amylases used now have poor thermal stability, etc. Aspect defects lead to an increase in industrial production costs. In order to adapt the main characteris...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/28
Inventor 王淑军陆兆新李富超吕明生陈丽邓祥元刘红飞
Owner HUAIHAI INST OF TECH
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