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Method for simultaneous high-yield production of cellulase and [beta]-glucosidase

A technology of glucosidase and cellulase, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low content of B-glucosidase, achieve the effects of reducing production costs, saving equipment investment and process energy consumption, and reducing costs

Inactive Publication Date: 2017-02-01
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for high-yield cellulase and β-glucosidase simultaneously, aiming at overcoming the problem that the content of B-glucosidase in the Trichoderma reesei cellulase system existing in the prior art is too low , provides a method for overexpressing the exogenous β-glucosidase gene aabgl1 under the strong promoter Ppdc1 in the presence of glucose, thereby simultaneously high-yielding cellulase and B-glucosidase with an inexpensive soluble inducer containing glucose

Method used

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  • Method for simultaneous high-yield production of cellulase and [beta]-glucosidase
  • Method for simultaneous high-yield production of cellulase and [beta]-glucosidase
  • Method for simultaneous high-yield production of cellulase and [beta]-glucosidase

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Experimental program
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Effect test

Embodiment 1

[0039]Embodiment 1, construction expresses the recombinant plasmid pCBHYGB-PB of exogenous β-glucosidase gene aabgl1

[0040] Using the plasmid pAN7-1 (see GenBank: Z32698.1 for the sequence) as a template, HygBF (5'-GGACTAGTCCTTGTATCTCTACACACAGGCTCA-3'SEQ ID No.10) and HygB (5'-GGACTAGTCCTTAATTAATCGAGTGGAGATGTGGAGTGGGCGC-3'SEQ ID No.11) as primers The hygromycin resistance gene was amplified, and the hygromycin expression cassette was connected to pCB301 (see GenBank: AF139061.1 for the sequence) Agrobacterium vector by using the Spe I restriction site, and the new vector obtained was named pCBHYGB. And using Trichoderma reesei strain Rut C30 (No. NRRL 11460) genomic DNA as a template, Ppdc1F (5'-CCCCCCTCGAGGTCGACGGTAGGACTTCCAGGGCTACTTG-3'SEQ ID No.4) and Ppdc1R (5'-CGGTCAGACTTCATGCCGGGCGTACGGATTGTGCTGTAGCTGCGCT-3'SEQ ID No.5 ) as primers to amplify the pdc1 promoter (SEQ ID No.1), Tpdc1F (5'-AGCGCAGCTACAGCACAATCCGTACGCCCGGCATGAAGTCTGACCG-3'SEQ ID No.6) and Tpdc1R (5'-AGGAATT...

Embodiment 2

[0041] Embodiment 2, using the method mediated by Agrobacterium tumefaciens to introduce the aabgl1 gene expression cassette into the Trichoderma reesei genome

[0042] The pCBHYGB-PB plasmid was transformed into Agrobacterium AGL-1 by electroporation method, and spread on the LB selective medium containing 100mg / L rifampicin and 100mg / L kanamycin (LB medium composition, g / L: 10NaCl, 10Tryptone, 5Yeast extract) plate, after culturing at 28°C for 2-4d, pick resistant single colonies for activation and preservation. In order to determine whether the transformation was successful, pick resistant colonies in the prepared PCR reaction system, use Pdcaabgl1F and Tpdc1R as primers for PCR reaction, and then take 5 μL of the amplified product for 1% agarose gel electrophoresis detection, the results are as follows figure 2 As shown, a band of about 3.5kb (3583bp) was obtained as expected.

[0043] Trichoderma reesei Rut C30 was inoculated in sporulation medium (3% malt extract powd...

Embodiment 3

[0049] Embodiment 3, use recombinant Trichoderma reesei to produce cellulase and β-glucosidase simultaneously

[0050] The fermentation method is as follows according to the high-yield cellulase method announced by Bai Fengwu et al. (201610309126.6): (1-3 steps are the same as the above-mentioned steps)

[0051] 1. Preparation of inducer: prepare 600g / L glucose solution, add 20CBU β-glucosidase produced by Xiasheng Industrial Group Co., Ltd. for each gram of glucose, incubate at pH 4.8 and 65°C for 72h, then boil the glucose solution for 5min to make After inactivation of β-glucosidase, the soluble inducer β-disaccharide and glucose mixture (MGD) can be prepared. It has been determined that MGD contains 410.3g / L of glucose, 60.5g / L of gentiobiose, 9.3g / L of cellobiose and 13.7g / L of sophorose.

[0052] 2. Incline culture: take a small amount of Trichoderrna reesei PB3 CGMCC No.12768 (recombinant Trichoderma reesei, according to the method for obtaining high-enzyme activity re...

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Abstract

The invention discloses a method for simultaneous high-yield production of cellulase and [beta]-glucosidase. The operating method for simultaneous high-yield production of the [beta]-glucosidase and the cellulase is implemented by the following steps: expressing polypeptide, which has a cellobiase activity, in trichoderma reesei so as to obtain a recombinant trichoderma reesei strain, and conducting fed-batch fermentation by taking a soluble inducer of a glycose transglycosylation product as a carbon source. According to the method provided by the invention, cultivation is conducted in a fermentation tank by virtue of a fed-batch fermentation technology for 156h, so that the activity of the [beta]-glucosidase in a recombinant strain fermentation broth exceeds 300CBU / mL, which is more than 60 times above that of an original strain, and meanwhile, the activity of the cellulase exceeds 50FPU / mL. With the application of the method provided by the invention, shortcoming in the prior art that the [beta]-glucosidase of a trichoderma reesei cellulase system is too low in enzymatic activity is overcome, the compounding cost of the cellulase is reduced and the enzymolysis efficiency of lignocellulose is improved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a method for simultaneously high-yielding cellulase and β-glucosidase, more specifically to a method for supplementing in batches with cheap soluble inducers in Trichoderma reesei Feed fermentation, simultaneously high-yielding method of cellulase and β-glucosidase. Background technique [0002] With the increasing energy crisis and environmental pollution, the bioconversion of lignocellulosic renewable resources represented by straw has become an important choice for the production of biofuels and bio-based chemicals to alleviate oil dependence. At present, the bioconversion efficiency based on lignocellulose needs to be improved, and the high production cost of cellulase is one of the important bottlenecks for its industrialization. Therefore, the high-efficiency and low-cost production of cellulase has attracted the attention of researchers fro...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N1/15C12N15/80C12R1/885
CPCC12N9/2437C12N9/2445C12N15/80C12N2800/60C12N2840/002C12Y302/01004C12Y302/01021C12Y302/01091
Inventor 赵心清白凤武李勇昊张晓月
Owner SHANGHAI JIAO TONG UNIV
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