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Constitutive acidic incision cellulase high-yield strain

An endo-cellulase and high-yielding strain technology, applied in the field of microbiology, can solve the problems of long fermentation period, large molecular weight, low enzyme activity, etc., and achieve the effects of good pH stability, short enzyme production period and high catalytic ability

Inactive Publication Date: 2012-09-05
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the cellulase produced by fungi are acidic extracellular enzymes with high enzyme production efficiency and complete enzyme system structure, but the cellulase produced by fungi are all compound enzymes, and the enzyme system structure is complex, the molecular weight is large, and it is difficult to separate and extract. The enzyme production process needs to be induced by adding cellulose substances, and the fermentation cycle is long, so it shows many shortcomings in the development of industrial enzymes
Although the cellulase produced by bacteria has a single component (that is, usually only contains one type of cellulase), the extraction steps can be simplified, but bacterial cellulase is mostly a neutral or alkaline intracellular enzyme, which is mostly adsorbed on the bacterial wall, and its Enzyme activity is generally low. In addition, most strains produce inducible endocellulase, which needs to be added with inducers, which is not suitable for large-scale industrial production.

Method used

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  • Constitutive acidic incision cellulase high-yield strain
  • Constitutive acidic incision cellulase high-yield strain
  • Constitutive acidic incision cellulase high-yield strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment illustrates the screening process of Bacillus subtilis LC:

[0039] Wherein the medium used for screening is:

[0040] Liquid medium formula (g / L): 3.0KH 2 PO 4 , 0.3MgSO 4 ·7H 2 O, 5.0 sucrose, 15.0 peptone, 30mL / L corn steep liquor.

[0041] Fermentation basal medium (g / L): 3.0KH 2 PO 4 , 0.3MgSO 4 ·7H 2 O, 5.0 yeast extract, 15.0 peptone.

[0042] Induction medium (g / L): Add 5g / L CMC-Na on the basis of the fermentation basal medium.

[0043] Congo red plate medium (g / L): 3.0KH 2 PO 4 , 0.3MgSO 4 ·7H 2 O, 5CMC-Na.

[0044] Take about 0.5 g of soil samples from the forest area of ​​Nanjing, add them to the liquid medium for enrichment culture (30°C, 180r / min), culture for 5-7 days and then transfer, and transfer three times in the same medium. Properly dilute the bacterial solution after enrichment culture, and the dilution gradient is determined according to the turbidity of the culture solution (105-107 times). Take 100 μL of the dilut...

Embodiment 2

[0049] This example illustrates the extraction of acidic constitutive endocellulase LC from Bacillus subtilis LC.

[0050] 1. Cell culture:

[0051] Solid medium formula (g / L): 1.0KH 2 PO 4 , 0.5MgSO 4 ·7H 2 O, 5.0 yeast extract, 10.0 peptone, the pH of the solid medium is 7.0-7.5;

[0052] Liquid medium formula (g / L): 3.0KH 2 PO 4 , 0.3MgSO 4 ·7H 2 O, 5.0 sucrose, 15.0 peptone, 30mL / L corn steep liquor, the pH of the seed liquid medium is 7.0, and the optimal initial pH of the fermentation medium is 5-5.5.

[0053] Inoculate Bacillus subtilis LC on solid medium, culture at 37°C for 10-12 hours, then transfer to 50mL / 250mL liquid seed medium, cultivate at 37°C and 200r / min for 10-12h, and then transfer to 2% inoculum Connect with 50mL / 250mL liquid fermentation medium and cultivate for 36h at 37°C and 200r / min.

[0054] 2. Treatment of extracellular product crude enzyme solution:

[0055] Freeze and centrifuge the fermentation broth at a speed of 10000r / min for 10min...

Embodiment 3

[0079] This example illustrates the relevant properties of the acidic constitutive endocellulase LC extracted from Bacillus subtilis LC:

[0080] (1) Basic kinetic constant K m and the maximum reaction speed V max Determination of

[0081] With different concentrations of CMC-Na as the substrate, under the conditions of the optimum catalytic temperature (65°C) and optimum pH (pH5.0), the enzymatic reaction rate was measured, and the K m value and V max , as attached image 3 Shown, where 1 / V=0.05607+1.32496×1 / S.

[0082] Michaelis constant K of endocellulase LC m 0.0423mg / mL, V max It is 135.85mg / min. It shows that the endocellulase LC has a stronger affinity to the substrate sodium carboxymethyl cellulose and has a strong catalytic ability.

[0083] (2) Enzymatic properties

[0084] 1. Determination of molecular weight of enzyme protein

[0085] Determined by SDS-PAGE electrophoresis, the results are attached figure 2 . Gel scanning was performed with GEL-DOC2000...

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Abstract

The invention relates to a constitutive acidic incision cellulase high-yield strain screened from a soil sample rich in plant cellulose, which has a category name of Bacillus subtilis LC and a preservation registration number of CCTCC No: M 208073. The screened Bacillus subtilis LC belongs to an uncommon constitutive cellulase high-yield strain. Compared with the prior bacterial strains of industrial enzymes, the constitutive acidic incision cellulase high-yield strain has the advantages of short fermentation enzyme production cycle, high enzyme activity and no need of induction in the enzyme production process; so the produced cellulase is the constitutive cellulase, and the constitutive acidic incision cellulase high-yield strain has the characteristic of being suitable for large-scale fermentation production.

Description

technical field [0001] The invention belongs to the technical field of microbiology, and in particular relates to a high-production strain of constitutive acid endocellulase. Background technique [0002] Cellulase is a general term for a group of enzymes that hydrolyze cellulose and its derivatives into glucose. A complete cellulase system usually consists of three types of enzymes that cooperate with each other to catalyze the hydrolysis of cellulose: endocellulase (CMCase), exocellulase and β-glucosidase. Endocellulase mainly acts on the non-crystalline region inside the cellulose molecule, randomly hydrolyzes the β-1,4-glucosidic bond, shortens the long-chain cellulose molecule, and produces a large amount of small molecular cellulose with non-reducing ends ; Exo-cellulase degrades small molecule cellulose to generate cellobiose and glucose; finally, β-glucosidase completely degrades cellobiose into glucose. Therefore, endocellulase plays the most critical role in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/42C12N15/56C12R1/125
Inventor 何冰芳欧阳平凯许婧柏中中吴斌
Owner NANJING UNIV OF TECH
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