High-yield cellulase trichoderma reesei engineering bacteria and preparing method and application thereof

Abstract

The invention discloses high-yield cellulase trichoderma reesei engineering bacteria which are obtained by guiding a gene bg11 of trichoderma reesei Rut-C30 into trichoderma reesei Rut-C30 by the adoption of the method of single enzyme digestion homologous recombination. The invention further discloses a construction method and application of the high-yield cellulase trichoderma reesei engineering bacteria. Compared with the prior art, extracellular beta-glucosidase generated by the engineering bacteria is increased by 17.3 times compared with that of the starting strain Rut-C30; meanwhile, endoglucanase, exoglucanase, filter paper enzyme activity and protein content are increased. In addition, the strain shows a high glucose endurance ability and a better saccharification ability.

Description

technical field

[0001] The invention belongs to the field of bioenergy and biotechnology, and in particular relates to a high-yield cellulase-producing Trichoderma reesei engineering bacterium and a preparation method and application thereof. Background technique

[0002] The oil energy crisis and the increasingly serious environmental pollution caused by the use of oil energy make people constantly pursue alternative renewable energy. Cellulose exists widely in nature and is a macromolecular polysaccharide polymer formed by connecting glucose residues through β-1,4 glycosidic bonds. It can be degraded into glucose by cellulase produced by fungi, and then used by microorganisms to produce bioenergy molecules, bio-based fine chemical products and pharmaceuticals. Cellulase is a complex enzyme mixture, and there are mainly three enzymes involved in cellulose degradation: endoglucanase, exoglucanase and β-glucosidase. First, endoglucanase degrades cellulose into cellooligosac...

Examples

Embodiment 1

[0059] The construction of the pBGL expression cassette comprises the following steps:

[0060] (1) The design of primers for bgl1 gene amplification is shown in Table 1. Using the cDNA of Trichoderma reesei Rut-C30 as a template, the primers were used to amplify the bgl1 gene. The amplification conditions are pre-denaturation: 95°C, 15s; denaturation: 95°C, 15s; annealing: 66°C, 30s; extension: 72°C, 3min; thorough extension: 72°C, 5min;

[0061] (2) The purified PCR product of the target gene was inserted into the XbaI restriction site of the plasmid pDht / sk by single-digestion homologous recombination to obtain the expression cassette pBGL, see figure 1 ;

[0062] (3) Adopting the method mediated by Agrobacterium to transfer the expression cassette into Trichoderma reesei spores The specific method is as follows:

[0063] Agrobacterium tumefaciens transformation:

[0064] a. Agrobacterium tumefaciens maintained at -70°C were incubated on ice for 10 minutes;

[0065] b....

Embodiment 2

[0074] The supernatants obtained from each strain of Trichoderma reesei cultured in cellulose medium shake flasks for 7 days were centrifuged at 4° C. at 8000 rpm for 15 minutes to collect the supernatants for the determination of cellulase activity. Cellulase activity assay method is as follows:

[0075] (1) Determination of β-glucosidase (pNPGase) enzyme activity: add 20 μl of appropriately diluted enzyme supernatant to 90 μl of 4 mM pNPG (50 mM NaCl, pH 5.0), incubate at 50°C for 10 min, draw 100 μl and add an equal volume of 2 %NaCO3, placed on a microtiter plate to read OD405 (Biochem-Tokyo 1999, 125(4):728-736).

[0076] (2) Determination of exoglucanase (pNPC) enzyme activity: 90μl of 4mM pNPC solution (0.05M NaAc, pH5.0, 1mg / ml 1,5-δ-gluconolactone) was added to 20μl of appropriate dilution factor Enzyme solution, incubate at 50°C for 30 minutes, draw 100 μl and add an equal volume of 2% NaCO3, place in a microtiter plate and read OD405 (Anal Biochem 1984,138(2):481-4...

Embodiment 3

[0080] The supernatants obtained from various strains of Trichoderma reesei cultured in cellulose medium with different concentrations of glucose in shake flasks for 7 days were centrifuged to collect the supernatants for the determination of cellulase activity. The cellulase activity assay method refers to Example 3, and the assay results are shown in FIG. 5 .

technical field

[0001] The invention belongs to the field of bioenergy and biotechnology, and in particular relates to a high-yield cellulase-producing Trichoderma reesei engineering bacterium and a preparation method and application thereof. Background technique

[0002] The oil energy crisis and the increasingly serious environmental pollution caused by the use of oil energy make people constantly pursue alternative renewable energy. Cellulose exists widely in nature and is a macromolecular polysaccharide polymer formed by connecting glucose residues through β-1,4 glycosidic bonds. It can be degraded into glucose by cellulase produced by fungi, and then used by microorganisms to produce bioenergy molecules, bio-based fine chemical products and pharmaceuticals. Cellulase is a complex enzyme mixture, and there are mainly three enzymes involved in cellulose degradation: endoglucanase, exoglucanase and β-glucosidase. First, endoglucanase degrades cellulose into cellooligosac...

Examples

Embodiment 1

[0059] The construction of the pBGL expression cassette comprises the following steps:

[0060] (1) The design of primers for bgl1 gene amplification is shown in Table 1. Using the cDNA of Trichoderma reesei Rut-C30 as a template, the primers were used to amplify the bgl1 gene. The amplification conditions are pre-denaturation: 95°C, 15s; denaturation: 95°C, 15s; annealing: 66°C, 30s; extension: 72°C, 3min; thorough extension: 72°C, 5min;

[0061] (2) The purified PCR product of the target gene was inserted into the XbaI restriction site of the plasmid pDht / sk by single-digestion homologous recombination to obtain the expression cassette pBGL, see figure 1 ;

[0062] (3) Adopting the method mediated by Agrobacterium to transfer the expression cassette into Trichoderma reesei spores The specific method is as follows:

[0063] Agrobacterium tumefaciens transformation:

[0064] a. Agrobacterium tumefaciens maintained at -70°C were incubated on ice for 10 minutes;

[0065] b....

Embodiment 2

[0074] The supernatants obtained from each strain of Trichoderma reesei cultured in cellulose medium shake flasks for 7 days were centrifuged at 4° C. at 8000 rpm for 15 minutes to collect the supernatants for the determination of cellulase activity. Cellulase activity assay method is as follows:

[0075] (1) Determination of β-glucosidase (pNPGase) enzyme activity: add 20 μl of appropriately diluted enzyme supernatant to 90 μl of 4 mM pNPG (50 mM NaCl, pH 5.0), incubate at 50°C for 10 min, draw 100 μl and add an equal volume of 2 %NaCO3, placed on a microtiter plate to read OD405 (Biochem-Tokyo 1999, 125(4):728-736).

[0076] (2) Determination of exoglucanase (pNPC) enzyme activity: 90μl of 4mM pNPC solution (0.05M NaAc, pH5.0, 1mg / ml 1,5-δ-gluconolactone) was added to 20μl of appropriate dilution factor Enzyme solution, incubate at 50°C for 30 minutes, draw 100 μl and add an equal volume of 2% NaCO3, place in a microtiter plate and read OD405 (Anal Biochem 1984,138(2):481-4...

Embodiment 3

[0080] The supernatants obtained from various strains of Trichoderma reesei cultured in cellulose medium with different concentrations of glucose in shake flasks for 7 days were centrifuged to collect the supernatants for the determination of cellulase activity. The cellulase activity assay method refers to Example 3, and the assay results are shown in FIG. 5 .

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