Cloning of beta-1,4-endo-glucanase gene and preparation of recombinase

Abstract

The invention provides a cloning method for complete mRNA and DNA sequences of beta-1,4-endo-glucanase gene from Aspergillus usamii E001. The nucleotide sequences of the complete mRNA and DNA are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. Bioinformatics analysis shows that the beta-1,4-endo-glucanase belongs to glycoside hydrolase family 12, is named Aus Cel12A, and has the amino acid sequence shown in SEQ ID NO:3; and the corresponding gene is named Aus cel12A. The invention also discloses a method for construction of Aus Cel12A engineering bacteria and high-efficiency expression and purification of recombinant Aus Cel12A. The most suitable operative temperature and pH of the prepared recombinant Aus Cel12A are respectively 60DEG C and 5.0; the recombinant Aus Cel12A is stable when the pH is 4.0-7.0 and the temperature is below 60DEG C, has higher industrial production potential and economic value, and lays a theoretical basis for researching other beta-endo-glucanase.

Description

technical field

[0001] The present invention relates to the cloning of the complete mRNA and DNA sequence of a β-1,4-endoglucanase gene derived from Aspergillus usamii (Aspergillus usamii) E001 strain, a glycoside hydrolase 12 family, β-1,4-endoglucanase gene The construction of the endoglucanase engineering bacteria and the high-efficiency expression and purification method of the recombinant β-1,4-endoglucanase belong to the technical field of bioengineering. Background technique

[0002] Cellulose is the most important structural component of plant cell walls and, together with hemicellulose, accounts for more than half of the earth's renewable organic carbon sources. Cellulase refers to the general term for a group of enzymes that can hydrolyze cellulose β-1,4 glucosidic bonds to generate cellobiose and glucose, mainly including endo-1,4-β-glucanase, EC 3.2 .1.4, referred to as EG), exo-glucanase (exo-1, 4-β-glucanase, EC 3.2.1.91, referred to as CBH) and β-glucosidase ...

Examples

Embodiment 1A

[0038] Cloning of Example 1 Aus cel12A 3' end mRNA sequence

[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Cel12A-3F1 as primers, and the reaction conditions were: 94°C for 2 min, 30 cycles (94°C 30s, 52°C for 30s, 72°C for 90s), 72°C for 10min; use M13 Primer M4 and Cel12A-3F2 as primers for the second round of PCR, and the reaction conditions are: 94°C for 2min, 30 cycles (94°C for 30s, 52°C 30s, 72°C 90s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-cel12A3′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 2A

[0040] Cloning of embodiment 2 Aus cel12A 5' end mRNA sequence

[0041] The first round of PCR was performed using the Outer Primer and Cel12A-5R1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10 min; the second round of PCR was performed using Inner Primer and Cel12A-5R2 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), and 72°C for 10 min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered and ligated with pUCm-T (pUCm-T-cel12A5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 3A

[0042] Example 3 Cloning of Aus cel12A 5' end promoter sequence

[0043] Using the processed A.usamii E001 genomic DNA as a template, the first round of PCR used T-PrimerF and Cel12A-5R1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C 60s), 72°C for 10min; the second round of PCR uses T-PrimerF and Cel12A-5R2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-cel12AP), transformed into JM109, and sent to Shanghai Sangon for sequencing.

technical field

[0001] The present invention relates to the cloning of the complete mRNA and DNA sequence of a β-1,4-endoglucanase gene derived from Aspergillus usamii (Aspergillus usamii) E001 strain, a glycoside hydrolase 12 family, β-1,4-endoglucanase gene The construction of the endoglucanase engineering bacteria and the high-efficiency expression and purification method of the recombinant β-1,4-endoglucanase belong to the technical field of bioengineering. Background technique

[0002] Cellulose is the most important structural component of plant cell walls and, together with hemicellulose, accounts for more than half of the earth's renewable organic carbon sources. Cellulase refers to the general term for a group of enzymes that can hydrolyze cellulose β-1,4 glucosidic bonds to generate cellobiose and glucose, mainly including endo-1,4-β-glucanase, EC 3.2 .1.4, referred to as EG), exo-glucanase (exo-1, 4-β-glucanase, EC 3.2.1.91, referred to as CBH) and β-glucosidase ...

Examples

Embodiment 1A

[0038] Cloning of Example 1 Aus cel12A 3' end mRNA sequence

[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Cel12A-3F1 as primers, and the reaction conditions were: 94°C for 2 min, 30 cycles (94°C 30s, 52°C for 30s, 72°C for 90s), 72°C for 10min; use M13 Primer M4 and Cel12A-3F2 as primers for the second round of PCR, and the reaction conditions are: 94°C for 2min, 30 cycles (94°C for 30s, 52°C 30s, 72°C 90s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-cel12A3′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 2A

[0040] Cloning of embodiment 2 Aus cel12A 5' end mRNA sequence

[0041] The first round of PCR was performed using the Outer Primer and Cel12A-5R1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10 min; the second round of PCR was performed using Inner Primer and Cel12A-5R2 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), and 72°C for 10 min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered and ligated with pUCm-T (pUCm-T-cel12A5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 3A

[0042] Example 3 Cloning of Aus cel12A 5' end promoter sequence

[0043] Using the processed A.usamii E001 genomic DNA as a template, the first round of PCR used T-PrimerF and Cel12A-5R1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C 60s), 72°C for 10min; the second round of PCR uses T-PrimerF and Cel12A-5R2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-cel12AP), transformed into JM109, and sent to Shanghai Sangon for sequencing.

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