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High-yield high-heat-stability bacillus licheniformis of beta-glucanase

A Bacillus licheniformis, thermal stability technology, applied in the field of microorganisms, to achieve high thermal stability and storage stability, a wide range of optimum pH value, and good thermal stability

Active Publication Date: 2014-05-28
HUNAN HONGYING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Previously, the high-quality β-glucanase in the market was almost monopolized by foreign enzyme preparation companies, although the enzyme activity of microbial β-glucanase reported in my country in recent years has reached or even exceeded the international level. , but obtaining β-glucanase with high activity and thermostability and its production strain is still a big problem to be solved in industrial application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Ultraviolet-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis Screening

[0056] (1) Preparation of bacterial suspension

[0057] Inoculate the β-10 single colonies grown after streaking on the plate into the seed medium, culture at 37°C for 48 hours at 100 r / min, take 1 mL of the culture medium, centrifuge, wash twice with normal saline, and resuspend in 9 mL of physiological in salt water.

[0058] The seed medium is: 0.5% of yeast powder, 1% of peptone, 1% of soluble starch and 1% of NaCl.

[0059] (2) Ultraviolet-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis

[0060] Place 3mL of bacterial suspension on a sterile plate, stir and irradiate for 100s under a UV lamp with a distance of 30cm and a power of 15w. The irradiated bacterial liquid was subjected to 10 -1 -10 -5 After gradient dilution, it was spread on a lithium chloride plate, and the bacterial solution without ultraviolet irradiation was diluted and spread on a plate as a control....

Embodiment 2

[0068] Example 2: Determination of β-glucanase activity

[0069] (1) Definition of enzyme activity unit: 1 mL of crude enzyme solution, at 50°C and pH 6.0, degrades 1 μmol of reducing sugar from a β-glucan solution with a concentration of 4 mg / ml in 1 minute, which is 1 enzyme activity unit (1U / ml)

[0070] (2) Enzyme Activity Determination Method: Determination by DNS method

[0071] ① Take 5 test tubes, numbered 1, 2, 3, 4, 5, and add 0.5ml of dextran solution respectively;

[0072] ② Add 0.5ml of the crude enzyme solution to be tested into test tubes 1, 2, and 3, and react in a water bath at 50°C for 15 minutes;

[0073] ③Add 0.5ml crude enzyme solution to be tested into test tubes 4 and 5;

[0074] ④ Add 3ml of DNS reagent to each of the 5 test tubes, shake well, and then boil in water for 5 minutes;

[0075] ⑤ After cooling in the water bath, measure the absorbance of No. 1, 2, and 3 at 540 nm with No. 4 and No. 5 test tubes as controls;

[0076] ⑥ Calculate the enzy...

Embodiment 3

[0077] Example 3: Determination of enzymatic properties

[0078] (1) The effect of temperature on the enzyme activity of β-glucanase

[0079] At 40, 50, 60, 70, 80, and 90°C, the activity of the obtained β-glucanase crude enzyme liquid was measured respectively, and the results showed that the enzyme had higher enzyme activity between 50-70°C, indicating that the The enzyme has a wide range of temperature adaptation, and its optimum temperature is 65°C. (2) Thermal stability of β-glucanase

[0080] The obtained crude enzyme solution was stored at 40, 50, 60, 70, 80, and 90°C for 10min, 30min, and 1h, and then the enzyme activity was measured at 50°C. The results showed that the enzyme was still able to Keep more than 80% of the enzyme activity, and keep more than 20% of the enzyme activity after being stored at 70°C for 10 minutes.

[0081] (3) Storage stability of β-glucanase

[0082] The obtained crude enzyme solution was stored at 4°C and room temperature for 1 day, 15 ...

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Abstract

The invention discloses a high-yield high-heat-stability bacillus licheniformis bacterial strain of beta-glucanase. The bacterial strain is obtained via the composite mutagenesis of ultraviolet-lithium chloride-sulphuric acid diethyl ester on an original bacterial strain. The bacillus licheniformis (Bacillus licheniformis) beta-lactamase 10-25 is preserved in China Center for Type Culture Collection, and the preservation number of the bacillus licheniformis is CCTCC NO: M2013538. The enzyme activity of the beta-glucanase produced by the bacterial strain is as high as 8000-10000 u / ml and is 2.5 times of that of the original bacterial strain. The enzyme activity of the beta-glucanase is higher than that of the existing beta-glucanase, the optimum pH value range of enzyme action is wide, the heat stability and the storage stability are both higher, and the bacillus licheniformis of beta-glucanase is especially suitable for industrialized demands with high reaction temperature and concurrent liquefaction technology and saccharification technology.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus licheniformis capable of producing high thermostable β-glucanase. Background technique [0002] β-glucan is a natural polysaccharide that usually exists in the cell walls of special types of bacteria, yeasts and fungi, or in the coating of higher plant seeds. β-glucan is a structural non-starch polysaccharide that forms the cell wall of grasses and is particularly abundant in the endosperm cell walls of barley, rye, sorghum, rice and millet. Unlike general sugars that are mainly combined with α-1,4-glycosidic bonds to form linear molecules, β-glucan in plant cell walls is a mixed β-(1,3), (1,4) glycoside The D-glucose polymer formed by the bond connection, so β-glucan can be dissolved in water with a large molecular weight, so it has the properties of high viscosity, high hydrophilicity, water swelling force and high water holding capacity...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C12N9/42C12R1/10
Inventor 李洪兵朱永明蒋珂毛强王小玲周俊
Owner HUNAN HONGYING BIOTECHNOLOGY CO LTD
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