Production method of recombinant multifunctional cellulase
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A cellulase, multifunctional technology, applied in the field of production of multifunctional cellulase, can solve the problems of high production cost, single component, complex cellulase production process, etc., and achieve the effect of difficult purification and low activity
Inactive Publication Date: 2011-06-15
SOUTH CHINA AGRI UNIV
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[0003] At present, the main production strains producing cellulase are Trichoderma, Penicillium and Aspergillus, and the production method is to obtain cellulase through solid fermentation or liquid fermentation of these filamentous fungi, because the cellulase produced by these filamentous fungi The composition is single, the activity of the enzyme is low in practical application, and it cannot effectively degrade cellulose (as mentioned above, the degradation of cellulose requires the synergy of multiple enzymes to complete), and the current production process of cellulase is complex and costly. high
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Embodiment 1
[0032] Prepare medium:
[0033] PDSA medium: Potato (peeled) 200g, glucose 20g, dipotassium hydrogen phosphate 3g, magnesium sulfate 1.5g, glucose 20g, agar 20g, distilled water to 1000mL, autoclaved for later use.
[0034] PDSB medium: no agar is added to the PDSA medium, and it is used after autoclaving.
[0035] Basic enzyme production medium: carbon source 20g, nitrogen source 4g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 0.46g, magnesium sulfate 1g, adjust the initial pH to 5.5, distilled water to 1000ml, high temperature sterilization.
[0036] Comparison of various production methods of recombinant multifunctional cellulase:
[0037] (1) According to Zhao Shuxian's master's thesis (multifunctional cellulase gene mfc Research on genetic transformation of Tremella spores, master thesis of South China Agricultural University, June 2008) to construct and obtain Tremella spore engineering strain ycLes3, inoculate it on a PDSA plate at 25°C for 4-5 d...
Embodiment 2
[0045] (1) Inoculate the ycLes3 strain prepared in Example 1 on a PDSA plate, incubate at 25°C for 4 days, pick a single colony and inoculate it in 100 mL of PDSB liquid medium, inoculate at 25°C, 180r / min shaking for 72h;
[0046] (2) Prepare bagasse medium, bagasse powder 20.31g / L, yeast extract 4.11 g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 0.46g / L, magnesium sulfate 1g / L, adjust the pH value 5.5; Divide into 250mL Erlenmeyer flasks, each bottle contains 100mL, and autoclave;
[0047] (3) In the ultra-clean work, each bottle was inoculated with 1mL of the bacteria cultured in step (1), and placed on a shaking table at 25°C after inoculation, and the shaking table speed was 180r / min.
[0048] (4) Terminate the culture after 4 days of culture, and collect the fermentation broth by centrifugation.
[0049] The PDSA solid medium formula wherein consists of the following parts by weight: 200 parts of potatoes, 20 parts of glucose, 3 parts of dipot...
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Abstract
The invention belongs to the field of microbial fermentation engineering, and relates to a production method of recombinant multifunctional cellulose. The production method is characterized by comprising the following steps: transforming a multifunctional cellulose gene into Tremella fuciformis spores to obtain a Tremella fuciformis spore engineered strain serving as a production strain; and carrying our shake flask fermentation by utilizing sugar cane bagasse powder as an optimal culture medium under optimized culture conditions, thus obtaining the recombinant multifunctional cellulase. The sugar cane bagasse culture medium has a pH value of 5.0-5.5 and comprises the following components: sugar cane bagasse powder 17-23 g / L, yeast extract 3.1-4.9 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.46 g / L and magnesium sulfate 1 g / L. The optimal culture conditions are as follows: the culture temperature is 25DEG C, the culture time is 4 days, and the rotation speed of a shaking table is 180r / min. The optimal enzyme yields are as follows: xylanase of enzyme activity 1.09*10<5>U / L, and carboxymethyl cellulase (CMCase) of enzyme activity 3.7*10<4>U / L. By utilizing the method provided by the invention, Tremella fuciformis spore engineered strain and cheap agricultural waste sugar cane bagasse can be effectively utilized to produce the recombinant multifunctional cellulose.
Description
technical field [0001] The invention belongs to the field of microbial fermentation engineering, and in particular relates to a method for producing multifunctional cellulase by using tremella spore engineering strains. A method for efficiently producing recombinant multifunctional cellulase using bagasse. Background technique [0002] Cellulose and hemicellulose are one of the most abundant renewable resources in nature and are the main components of plant cell walls. Every year, the earth produces hundreds of millions of tons of plant dry matter (polysaccharides) through photosynthesis, the main components of which are cellulose and hemicellulose. The enzymatic degradation of cellulose and hemicellulose is currently recognized as the most effective and clean conversion method. Its biodegradation requires the action of cellulase. Cellulase is a complex enzyme system, mainly including endo-β-1,4-D-glucanase (E.C. 3.2.1.4), exo-β-1,4-D-glucanase (E.C. 3.2.9.11) and beta-g...
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