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High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof

A technology of Bacillus subtilis and Bacillus, applied in the direction of enzymes, bacteria, enzymes, etc., can solve the problems of high production cost of cellulase, long production cycle of fungi, and long time consumption, and achieve a wide range of temperature and pH stability, easy to use The effect of pure culture and stable fermentation performance

Inactive Publication Date: 2021-01-22
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high production cost of cellulase is mainly due to two reasons: First, the enzyme activity produced by the production strain is low, which is also the main constraint factor limiting the large-scale production of cellulase
Secondly, the current cellulase-producing bacteria are mainly fungi, and the production cycle of fungi is longer, generally more than 6 days, which takes a long time and is inefficient

Method used

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  • High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof
  • High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof
  • High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Enrichment culture:

[0042] Add 1g of soil sample to 50ml enrichment medium, the composition of enrichment medium is: 1-2.5g / L KH 2 PO 4 , 1.2-1.8g / L (NH4) 2 SO 4 , 0.3-0.6g / L MgSO 4 .7H 2 O, 0.1-0.4g / L CaCl 2 , 0.001-0.005g / LFeSO 4 .7H 2 O, 0.5-1.5g / L tryptone, 5-10g / L CMC-Na. Cultivate at 37° C. and 200 rpm for 48 hours. After the end of the cultivation, take 1 ml of the culture solution and re-cultivate in the same condition in the fresh enrichment medium, and repeat three times.

[0043] Primary screening:

[0044] CMC-Na plate experiment

[0045] Take 1ml of the enriched culture solution and spread it on the CMC-Na plate after appropriate dilution. After staining with Congo red and NaCl decolorization, a single colony with a hydrolysis circle is obtained, and the enrichment culture is carried out, and the sample is again spotted on a new CMC-Na plate. To obtain accurate hydrolysis zone diameter / colony diameter data, such as figure 1 shown. The composi...

Embodiment 2

[0053] Design primers for 16S rRNA sequence amplification, blast Z2 16S rRNA sequence and perform phylogenetic tree analysis, the results show that Z2 is Bacillus subtilis, such as Figure 4 shown.

Embodiment 3

[0055] Effect of pH and temperature on enzyme activity:

[0056] Take an appropriate amount of enzyme solution and the same volume of buffer solution with a pH range between 4.0 and 10.0 and incubate overnight at 50°C, then use 2-5% CMC-Na as a substrate to detect enzyme activity, and obtain the optimal pH of 7 . Among them, Citrate buffer (pH3.0–5.0), Tris-HCl buffer (pH 6.0–8.0) and Glycine-NaOH buffer (pH 9.0–10.0). Adjust the pH to 7, heat in a water bath at 30°C-80°C overnight, and then use 2-5% CMC-Na as a substrate to detect the enzyme activity, and the optimal temperature is 50°C. The effect of pH on CMC enzyme activity is as follows: Figure 5 As shown, the effect of temperature on CMC enzyme activity is as Figure 6 shown.

[0057] Effects of pH and Temperature on Enzyme Activity Stability

[0058] Take an appropriate amount of enzyme solution and the same volume of buffer solution with a pH value ranging from 4.0 to 10.0 and incubate at 50°C for 30 minutes, the...

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Abstract

The invention discloses a high-temperature-resistant high-yield cellulase bacillus subtilis and application thereof. A cellulase production strain is separated from surface soil of a corn straw pile in a Jiangjin area of Chongqing, and the preservation number of the bacillus subtilis is CCTCC NO: M 2020002; a strain Z2 with the best enzyme production performance is finally obtained through enrichment culture, a CMC-Na plate experiment, a lignin plate experiment and an enzyme production experiment, and the obtained strain is subjected to 16S rRNA sequence comparison and identification to determine that the strain is bacillus subtilis Z2. By optimizing the culture medium and the culture conditions, the filter paper enzyme activity, the CMC enzyme activity and the beta-glucosidase activity under the optimal enzyme production conditions of 0.800 U / ml, 5.20 U / ml and 2.07 U / ml are obtained respectively. Cellulase produced by the strain has relatively high activity and stability under the conditions of a buffer solution with the pH value ranging from 4.0 to 10.0 and the temperature of 30 DEG C and 80 DEG C. Cellulase produced by the strain is mixed with commercial xylanase, then a saccharification experiment is carried out on pretreated straw stalks, and 84.27 mg / ml of total reducing sugar can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-temperature-resistant cellulase-producing Bacillus subtilis and an application thereof. Background technique [0002] Plant-derived lignocellulose is a renewable natural carbohydrate polymer. Effective utilization of abundant natural biomass has been a hot topic in recent years. Cellulase is a general term for a class of multicomponent enzymes that can convert cellulose into fermentable sugars. The main components of cellulase are exoglucanase, endoglucanase and β-glucosidase, and other auxiliary proteins, such as cellobiose dehydrogenase and swellin. Cellulase has a good application prospect in food, fuel ethanol preparation, feed processing and other industries. [0003] Cellulase-producing bacteria include bacteria, fungi, and actinomycetes. Bacteria are an efficient source of industrially important enzymes for converting cellulosic biomass due to their high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12R1/125
CPCC12N1/20C12N9/2437C12R2001/125C12N1/205
Inventor 王永忠刘帅全林丁柯吉婕莉刘乙
Owner CHONGQING UNIV
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