Strain for producing heat-resistant alkaline cellulase with high yield and production method thereof

A cellulase and alkaline technology, which is applied in the production field of Bacillus with high heat-resistant alkaline cellulase, can solve the problems of low enzyme activity, low fermentation level, unreasonable enzyme system composition, etc., and achieve pH value Wide application range, single component, good stability

Inactive Publication Date: 2021-02-26
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems in the production of alkaline cellulase, such as low enzyme activity, low fermentation level, unreasonable enzyme system composition, etc., which seriously limit the industrial application of alkaline cellulase

Method used

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  • Strain for producing heat-resistant alkaline cellulase with high yield and production method thereof
  • Strain for producing heat-resistant alkaline cellulase with high yield and production method thereof
  • Strain for producing heat-resistant alkaline cellulase with high yield and production method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The mutagenesis of embodiment 1 bacterial strain

[0037] Inoculate the starting strain into the seed medium, culture at 37°C, 200r / min, for 24h, and obtain the suspension of the bacteria to be mutated.

[0038] Put the bacterial suspension in a sterile centrifuge tube, centrifuge at 8000r / min for 10min to separate the precipitated bacteria, discard the supernatant, wash with normal saline-precipitated bacteria twice, then resuspend the bacteria with buffer, and add NTG Mother liquor, so that the final concentration of NTG is 0.5g / L. Under the condition of 37°C and 100r / min reciprocating shaking, mutagenesis was carried out for 20min. At the same time, do a control experiment, replace NTG mother solution with buffer solution, and other treatments are the same.

[0039] After the mutagenesis is over, centrifuge and wash 3 times with buffer, resuspend the bacteria, take 200 μL of the bacterial suspension for gradient dilution, take 100 μL and spread it on the screening ...

Embodiment 2

[0042] The screening of embodiment 2 bacterial strains

[0043] High-yielding strains were screened by combining transparent circle screening with conventional viability assays.

[0044] Primary screening: isolate and screen vigorously growing colonies, transfer them to solid screening plate B (medium B) with a toothpick, and culture them upside down at 37°C. After the bacteria grow out, cover the culture dish with an appropriate amount of 1 mg / mL Congo red solution and stain Discard the dye solution after 15 minutes, add an appropriate amount of 1mol / L NaCl solution for washing, and pour off the NaCl solution after 15 minutes. A clear transparent circle will appear around the colony producing cellulase (CMC enzyme), and the size of the transparent circle can reflect the level of enzyme activity. The strains with large transparent circles were screened and purified by streaking on plates. After repeated 3 times, a single colony was picked and inoculated into slant medium A, c...

Embodiment 3

[0051] Example 3 Stability Passage Test of High Yield Bacterial Strain AC-77

[0052] The mutant strain AC-77 was subcultured, and the enzyme activity of the shake flask fermentation of the first-generation strain was taken as 100%, and the relative enzyme activity was calculated. It can be seen from Table 1 that the enzyme production ability of the strain has not changed much after 10 consecutive passages, and has good passage stability.

[0053] Table 1. Results of genetic stability experiments of strain AC-77

[0054] Shake Flask Enzyme Activity (U / mL) Relative enzyme activity (%) N1 76.1 100 N2 77.1 101 N3 75.1 99 N4 76.6 101 N5 72.3 95 N6 78.0 102 N7 78.0 102 N8 73.3 96 N9 73.2 96 N10 79.9 105

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Abstract

The invention relates to the technical field of biology, and particularly relates to a strain for producing heat-resistant alkaline cellulase with high yield and a production method thereof. The strain is particularly Bacillus sp. AC-77, and the preservation number is CGMCC No. 20674. The strain is obtained through NTG mutagenesis, the average shake flask fermentation activity of produced alkalinecellulase is 76U / mL, the fermentation tank enzyme activity is 150U / mL or above, the produced cellulase has high heat resistance and alkali resistance, meanwhile has higher alkali-resistant endonuclease activity and lower filter paper and exocellulase activity, and the filter paper enzyme activity, the exocellulase activity and the beta-glucosidase activity are all low. The strain for producing heat-resistant alkaline cellulase with high yield can be widely applied to neutral and alkaline industrial production and has good application prospects in the industries of pulping and papermaking, washing finishing of natural cellulose fabrics, detergents and the like.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a high-yielding heat-resistant alkaline cellulase bacillus and a production method. Background technique: [0002] Cellulose is the most widely distributed and abundant renewable organic resource on earth. Natural cellulose is composed of regular and neat crystalline regions and relatively irregular and loose non-crystalline regions (amorphous regions). Microorganisms can produce a large number of enzymes to degrade and modify cellulose in nature, converting natural cellulose into Feed, chemical and other raw materials. Among them, cellulase is a general term for a class of enzymes that can hydrolyze cellulose into reducing sugars, and there is a synergistic effect between enzymes. [0003] Alkaline cellulase is a component of the cellulase system, that is, carboxymethyl cellulase (CarboxymethylCellulase, CMCase), also known as endonuclease, was first obtained from the culture of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12R1/07
CPCC12N1/20C12N9/2437C12R2001/07C12N1/205
Inventor 王克芬刘金巍王金余刘文龙王兴吉张杰
Owner SHANDONG LONGKETE ENZYME PREPARATION
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