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A low-temperature cellulase and xylanase-producing Bacillus cereus

A technology of Bacillus cereus and cellulase, which is applied in the field of microbial engineering, can solve the problems of discounted enzyme activity, low enzyme activity of enzyme production, and few studies on enzyme production of Bacillus, achieving good enzyme activity and high enzyme activity , Excellent pH tolerance effect

Active Publication Date: 2022-07-08
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are a variety of microorganisms in the natural environment, but most of them are greatly reduced due to the limitation of ambient temperature or bacterial growth rate. Usually, the temperature is low, the growth of microorganisms will be inhibited, and the enzyme activity is low. Especially in the cold environment in the north for a long time, there are few studies on the production of enzymes for the survival of Bacillus

Method used

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  • A low-temperature cellulase and xylanase-producing Bacillus cereus
  • A low-temperature cellulase and xylanase-producing Bacillus cereus
  • A low-temperature cellulase and xylanase-producing Bacillus cereus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Screening and identification of original strain TK-1

[0022] (1) Isolation of strains

[0023] (1) Collect 5 g soil samples from the forest area of ​​Changbai Mountain Nature Reserve, dissolve them in 100 mL of prepared salt solution, and incubate them with shaking at 10°C and 180 rpm for 2 to 4 hours. The salt solution is 0.006 mM FeSO 4 ·7H 2 O; 0.01 mM CaCO 3 ·7H 2 O; 0.08mM MgSO 4 ·7H 2 O; 0.07mM MnSO 4 ·7H 2 O and 0.006mM ZnSO 4 ·7H 2 O composition.

[0024] (2) Dilute 1 mL of the mixed soil sample salt solution with different gradients (10-2, 10-3, 10-4), take 200 ul of the diluted solution and spread it on the R2a solid plate, and place it at 10 °C for constant temperature incubation Incubate for 7-10 days, and the fixed plate contains 0.5g yeast extract powder, 0.5g peptone, 0.5g casein hydrolyzate, 0.5g glucose, 0.5g soluble starch, 0.3g potassium dihydrogen phosphate, 0.024g anhydrous Magnesium sulfate, 0.3 g sodium pyruvate, 15.0 g agar...

Embodiment 2

[0041] Example 2: Mutagenesis of original strain TK-1

[0042] Using ARTP mutagenesis, the mutagenesis steps are as follows:

[0043]The power is 100W, the mutagenesis working gas is helium, the gas flow rate is 12s / m, and the distance is 3mm. Incubate in an incubator. Take 1 mL of bacterial liquid cultured to the logarithmic phase (OD600 is between 0.6 and 0.8) in a 1.5 ml centrifuge tube, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and resuspend the bacterial cells with physiological saline twice to obtain a cell suspension. Dilute to adjust cell number at 10 6 -10 8 pcs / ml.

[0044] Take 10uL of cell suspension and spread it evenly on the surface of the metal slide (sterilized), and adjust the parameters of the ARTP instrument:

[0045] The treated slides were transferred to a centrifuge tube containing 1 mL of physiological saline, shaken for 1 min, and then placed in a shaking incubator (15° C., 22 rpm) for 1 hour of shaking culture.

[0046] Then t...

Embodiment 3

[0047] Example 3: Growth and enzyme production analysis of Bacillus cereus TK-2

[0048] (1) Analysis of growth characteristics of Bacillus cereus TK-1 and TK-2

[0049] (1) The mutant strain TK1 and mutant strain TK-2, which grow well at low temperature and produce cellulase and xylanase, were inoculated into LB liquid medium, respectively, at a rotating speed of 180 rpm, at different temperatures (10 , 15, 20, 25, 37°C) for shaking culture, sampling at fixed points, and measuring the OD of the bacterial liquid 600 value of .

[0050] (2) Analysis of enzyme production characteristics of Bacillus cereus TK-1 and TK-2

[0051] (1) Inoculate the logarithmic phase bacterial liquid into the fermentation enzyme production medium (ammonium sulfate 4g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.1g / L, yeast extract 10g / L, peptone 1g / L, xylan / CMC-Na 1.5%), the inoculation volume was 200uL, the rotation speed was 180rpm, and the shaking culture was carr...

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Abstract

A kind of Bacillus cereus that produces cellulase and xylanase at low temperature, which is preserved in the China Type Culture Collection and Management Center, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the classification name is waxy Bacillus ( Bacillus cereus ) TK‑2, the deposit number is CCTCC NO: M2021433, and the deposit date is April 22, 2021. Bacillus cereus TK-2 has low temperature tolerance and is suitable for growth and enzyme production at lower temperatures. In particular, the enzyme activity of xylan produced at a low temperature of 15 °C reaches 2.3 times that at 37 °C. The enzymatic activity of carbohydrase can reach 60.57IU / mL, and the enzymatic activity of cellulase (endo-cellulase) is 28.2IU / mL. The produced xylanase and cellulase still have the same properties at lower catalytic temperature. High enzymatic activity, excellent pH tolerance, and good enzymatic activity in weakly acidic, neutral and alkaline environments.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to a Bacillus cereus that produces cellulase and xylanase at low temperature. Background technique [0002] Xylan (xylan) is a polypentacarbon sugar, the main component of hemicellulose in plant cell walls, and another abundant regenerative polysaccharide in nature besides cellulose. Xylanase can convert xylan into xylo-oligosaccharides and xylose, and has broad application prospects in agriculture, energy, feed, paper, food and other fields. There are various sources of xylanase, among which the xylanase produced by microorganisms is one of the main sources. Currently, the industrial application of xylanase is restricted due to the limited types of xylanase-producing strains, the high production cost, and the inefficient and stable enzyme-producing ability of the strains. Therefore, it is of great significance to develop a new strain of xylanase with high activity a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/42C12N9/24C12R1/085
CPCC12N9/2437C12N9/248C12Y302/01004Y02E50/10
Inventor 张斯童沈丽君包昌杰陈欢王刚孙旸陈光
Owner JILIN AGRICULTURAL UNIV
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