Bacteria and breeding method and method for producing cellulase thereof

A cellulase and cellulose technology, applied in microorganism-based methods, methods using microorganisms, bacteria, etc., can solve problems such as unfavorable separation and purification of cellulase, unfavorable application, and short cycle of bacterial cellulase production.

Inactive Publication Date: 2012-07-18
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the long production cycle of fungal cellulase; the specific activity of cellulase is low; the secreted cellulase is mainly acid cellulase, which is not conducive to the application in textile industry and other aspects. These shortcomings are to a certain extent Both limit the application of fungal cellulase
[0005] The cellulase expressed by most bacteria is generally not secreted outside the cell, which is not conducive to the separation and purification of cellulase, and the yield of cellulase is lower than that produced by fungi, but many cellulose-degrading bacteria, especially rumen anaerobic bacteria produce The specific activity of the cellulase is high, and the cycle of cellulase produced by bacteria is short. Generally, the fermentation can be completed in about 2 days. The neutral cellulase and alkaline cellulase produced by bacteria have special industrial applications, so bacteria The application of cellulase is more and more extensive

Method used

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  • Bacteria and breeding method and method for producing cellulase thereof
  • Bacteria and breeding method and method for producing cellulase thereof
  • Bacteria and breeding method and method for producing cellulase thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Isolation of the cellulose-degrading microorganism Cellulomonas moist from bovine rumen fluid ( Cellulomonas uda ) NJ0807

[0036] 1 Sampling of bovine rumen fluid

[0037] The stomach of buffalo buffalo just slaughtered in a slaughterhouse in Anhui was taken out, the rumen juice was quickly put into a sterile bottle sterilized by high temperature in advance, and the bottle containing the cow rumen juice was quickly transported to the laboratory in a low temperature environment.

[0038] 2 Enrichment culture of cellulose degrading microorganisms

[0039] Cellulose-degrading microorganisms in bovine rumen juice were enriched and cultured in an enriched medium with cellulose as the only carbon source. The composition of the enrichment medium is 0.5% CMC, 0.5% filter paper, 0.2% NaNO 3 , 0.1% K 2 HPO 4 , 0.05% KCl, 0.05% MgSO 4 ·7H 2 O, 0.0001% FeSO 4 , pH natural. The medium was steam sterilized at 121°C for 20 min. Bovine rumen juice was inoculated into a 300mL...

Embodiment 2

[0048] Production of cellulase using the above strains

[0049] The strain Cellulomonas moist ( Cellulomonas uda ) NJ0807 was streak-inoculated into LB solid medium, cultured at 30 °C for 3 days, and then inoculated into liquid seed medium. The composition of the seed medium was 0.5% yeast extract, 0.5% sucrose, 0.4% NaCl, 0.1% K 2 HPO 4 , 0.05% MgSO 4 ·7H 2 O, adjust the initial pH of the medium to 7.5, and sterilize at 121 °C for 20 min. After the bacteria were cultured in the seed medium at 28°C and 180 rpm for 30 hours, the concentration of bacteria accumulated in a large amount, and the seed liquid was inoculated into a 1 L Erlenmeyer flask containing 400 mL of liquid fermentation medium at a 5% (v / v) inoculation amount , the composition of the liquid fermentation medium was 1% peptone, 1% CMC, 0.5% sucrose, 0.4% NaCl, 0.1% K 2 HPO 4 , 0.05% MgSO 4 ·7H 2 O, adjust the initial pH of the medium to 7.5, and sterilize at 121 °C for 20 min. After culturing for 2 days...

Embodiment 3

[0051] Purification of cellulase

[0052] Dissolve the ethanol-precipitated crude enzyme liquid product in an appropriate volume of 20 mM Tris-HCl (pH 8.8) buffer and purify using an anion exchange chromatography Q Sepharose Fast Flow fast protein liquid chromatography (FPLC) purification system using XK 16 Preparative column, ion exchange resin using 20 mmol L -1 Equilibrate with Tris-HCl (pH 8.8) buffer, wash and equilibrate with 10 times column volume of equilibration buffer, add the dissolved crude enzyme solution, the total protein amount loaded is about 10% of the column adsorption capacity. Gradient elution was used for elution, and the NaCl concentration rose to 0.4 mol L within 8 column volumes -1 , with a flow rate of 2 mL min -1 . Collect in different tubes, collect 2 mL in each tube, measure the cellulase activity of the collected parts and combine the active fragments. This purification step resulted in two main cellulase components, in Figure 4 Correspondi...

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Abstract

Cellulose producing bacteria are named as cellulomonas uda NJ0807, a bacterial strain is preserved in a China microorganism culture preservation management committee common microorganism center on June 29th,2011, and a preservation registration number is CGMCC No.5022. A login number of a 16S r deoxyribonucleic acid (rDNA) sequence in a GenBank is HM034815. The bacteria are novel wild cellulose degradation bacteria and are screened from rumen fluid of a cow. The bacteria are placed in a medium of cellulose resources of microcrystalline cellulose, carboxymethylcellulose and rice husk and the like and can produce cellulase components through liquid shake-flask fermentation. The invention provides a purifying method for main components of the cellulase simultaneously, and three main cellulase components are purified through the purifying method. The method for preparing the cellulase through bacteria fermentation belongs to the field of microorganism manufacture and has potential application prospects.

Description

technical field [0001] The invention belongs to the field of microbial production, and relates to a bacterium, a breeding method, and a method for producing cellulase. Background technique [0002] Cellulose is the most widely distributed and abundant carbohydrate on the earth, and it is also the largest renewable resource on the earth. It is estimated that the annual content of cellulose in plants produced by photosynthesis is 8.5×10 10 tons, equivalent to four times the world's total annual energy consumption (Yung-Chung Lo et al., 2009). The effective use of cellulose, a huge renewable resource, has great practical significance for solving environmental pollution, food shortage, and energy crisis, and can create considerable economic and social benefits (S. M. Read and T. Bacic, 2002). Bioconversion of cellulose by microorganisms and their metabolites can be widely used to produce sustainable biosourced products to replace depleted fossil fuels (Angenent LT et al., 2004;...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/22C12N9/42C12R1/01
Inventor杨树林郑德严菊芬
OwnerNANJING UNIV OF SCI & TECH