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Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof

A kind of technology of Saccharomyces saccharomyces and purification method, which is applied in the field of applied microorganisms, and can solve the problems of no high-temperature actinomycetes and the like

Inactive Publication Date: 2010-01-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no report on the thermostable actinomycetes RHA1 strain, and the heat-resistant α-amylase obtained therefrom and its purification method

Method used

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  • Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof
  • Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof
  • Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Isolation, screening and identification of Saccharomyces saccharomyces hyperthermia strain RHA1:

[0035] Thermoactinomycetes RHA1 strain, isolated from Tengchong Hot Spring. Strain selection medium is (g / L): (NH 4 ) 2 SO 4 , 1.3; peptone, 1; soluble starch, 4; KH 2 PO 4 , 0.276; MgSO 4 ·7H 2 O, 0.25; yeast extract, 1; Na 2 MoO 4 2H 2 O, 0.000025; CuSO 4 , 0.000016; MnSO 4 ·H 2 O, 0.0022; H 3 BO 3 , 0.0005; ZnSO 4 ·7H 2 O, 0.0005; CoCl 2 ·6H 2 O, 0.000046; CaSO 4 ·H 2 O, 0.06; FeSO 4 , 0.099; pH7.5. The culture conditions are temperature 50° C., shaking culture at 150 rpm, and the culture time is about 18 hours.

[0036] The bacterial system identification method was used to identify the RHA1 strain of Saccharomyces thermoactinoides. The morphological characteristics of the bacteria are as follows: the white cloth is transparent, wrinkled, dry on the surface, and the front and back colors are different, Gram-positive bacteria. Observed by electro...

Embodiment 2

[0038] Purification of α-amylase:

[0039] (1) Concentration by ultrafiltration: the fermentation and culture conditions of the hyperthermic actinomycetes RHA1 strain are temperature 50° C., pH 7.5, and the culture time is about 18 hours. Centrifuge the fermentation broth, take the supernatant, and use a 10KDa ultrafiltration membrane bag for ultrafiltration and concentration.

[0040] (2) Ammonium sulfate precipitation: Add ammonium sulfate to the concentrated liquid to 40% saturation, centrifuge to take the supernatant, continue to add ammonium sulfate to 60% saturation, centrifuge to discard the supernatant, and finally use pH7.0 phosphate buffer for precipitation The solution was dissolved to make a crude enzyme solution.

[0041] (3) Superdex 7510 / 300 gel filtration chromatography: centrifuge the obtained crude enzyme solution, concentrate the supernatant, and load it on a Superdex 7510 / 300 gel pre-equilibrated with 50mM, pH7.0 phosphate buffer Filter the chromatographi...

Embodiment 3

[0045] Characteristics of α-amylase:

[0046] ①Optimum enzyme activity temperature

[0047] Add enzyme liquid to phosphate buffer (50mM phosphate buffer, pH 7.0), use soluble starch as substrate, under the condition that the substrate concentration is 1%, measure the amylase produced by RHA1 strain at 28-98 Enzyme activity at different temperatures in the °C range. The result is as figure 1 As shown, it shows that the optimum enzyme activity temperature of the α-amylase is 37°C.

[0048] ②Thermal stability

[0049] Add the enzyme solution to phosphate buffer (50mM phosphate buffer, pH 7.0), keep it warm in constant temperature water baths at different temperatures, take samples at different times (0-90min, sampling interval is 15min), and then follow the standard Enzyme activity assay method to measure enzyme activity. The effect of temperature on enzyme stability is expressed as the percentage of remaining enzyme activity in the original enzyme activity after a certain p...

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Abstract

The invention discloses a method for preparing high-temperature alpha-amylase by a heat resistant high-temperature laceyella sacchari RHA1 virus strain and a purification method thereof. The high-temperature laceyella sacchari RHA1 virus strain is white and transparent, has corrugations, dry surfaces, different positive colors and negative colors, ball shapes, laceyella sacchari gram-positive bacterium, no flagella or no pilus; and a large number of mycelium is produced when culturing liquid. The method for preparing the amylase by the high-temperature laceyella sacchari RHA1 comprises the following steps: carrying out ultrafilter concentration and ammonium sulfate precipitation on a fermentation supernatant through an ultrafilter envelope of 1KD to obtain crude enzyme liquid, and filtering by Superdex 75 10 / 300 gelatin to obtain pure heat resistant alpha-amylase. The molecular weight of the alpha-amylase obtained in the invention is only 11.9 KDa, and the alpha-amylase is a monomer protein and is an amylase with the minimal molecular weight reported recently. The alpha-amylase hydrolyzes amylose and soluble starch to obtain maltose and maltotriose, can resist high temperature and has wide action temperature range and application values in food processing, medicines, detergents and the like.

Description

Technical field: [0001] The invention belongs to the field of applied microorganisms, in particular, it relates to a strain of Laceyella sacchari (Laceyella sacchari) RHA1, a heat-resistant α-amylase obtained therefrom and a purification method thereof. Background technique: [0002] α-amylase, also known as liquefied amylase, can hydrolyze β-1,4-glucosidic bonds in starch polysaccharide molecules to produce monosaccharides and oligosaccharides. It has been used in many fields, for example, in fermentation broth for saccharification of cereals and potatoes, in the textile industry as a starch paste remover, in the pharmaceutical industry as a digestive aid, and in the food industry Used to make thick malt syrup. [0003] Since amylase generally needs to gelatinize starch at high temperature during liquefaction and saccharification, the conversion of starch requires heat-stable amylase. Thermostable α-amylase has good thermal stability and has broad application prospects in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/28C12R1/01
Inventor 林连兵魏云林季秀玲井申荣李秋鹏洪伟陈波陆月情熊学权
Owner KUNMING UNIV OF SCI & TECH
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