A kind of mannanase and its application
A technology of mannanase and gene, applied in the field of microbial engineering, to achieve the effects of improving utilization rate, reducing material-to-weight ratio, saving food resources and breeding costs
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Embodiment 1
[0013] Example 1 Cloning of Mannanase Gene
[0014] Pick a single colony of fresh Bacillus subtilis CGMCC1.897 (purchased from the China General Microorganism Culture Collection Management Center) in 5 mL LB liquid medium, and shake it at 200 rpm at 37 ° C overnight, and follow the Tiangen Bacterial Genome Extraction Kit (day Root Biochemical Technology Co., Ltd.) to extract chromosomes.
[0015] Primers were designed according to the homologous sequence of mannanase on NCBI, and the chromosome of Bacillus subtilis CGMCC1.897 was used as a template, and the upstream and downstream primers were used to amplify. Anneal for 40s, extend at 72°C for 60s, and after 30 cycles, extend at 72°C for 10min. The PCR amplification product was recovered by the gel recovery kit, and the product was named as Man-1 .
[0016] Will Man-1 Cloned into the pMD18-T vector, the plasmid pT-Man-1 was constructed, and sent to Beijing Huada Gene Sequencing Center for sequencing. Man-1 The gene se...
Embodiment 2
[0017] Example 2 Construction of expression vector
[0018] The plasmid pT-Man-1 was used as a template and primers were used for PCR amplification. The PCR amplification conditions were 95°C for 4min; 94°C for 30S; 55°C for 40S; 72°C for 1.2min for 30 cycles; 72°C for 7min. Gel recovery amplification products; Eco RI and Not I double digestion. The expression plasmid pPIC9K was also performed Eco RI and Not I double digestion; use T4 ligase to ligate the above double digestion product at 4°C overnight. Finally, the ligated product was introduced into E. coli BL21. The expression plasmid of the corresponding positive clone was named pPIC-Man-1.
Embodiment 3
[0019] Example 3 Construction of Pichia pastoris engineering bacteria
[0020] The expression plasmid pPIC-Man-1 was used Sal After identification by enzyme digestion and electrophoresis, the DNA concentration was determined by ethanol precipitation and concentration, and the plasmid fragment was diluted at a concentration of 3 μg / μL and stored for later use. Prepare Pichia pastoris GS115 electrotransformation competent cells, and finally resuspend in 1 mL pre-cooled electrophoresis buffer (containing 1mM MgCl 2 , 10mM HEPES, 250mM sucrose, pH 7.8). Add 5 μL linearized recombinant plasmid to 80 μL competent cells; electroporation (conditions: 1500V, 200Ω, 25μF); finally spread on MM plate (MM medium component: 1.34%YNB, 4×10 -5 % biotin, 0.5% methanol), select a recombinant strain, named Pichia Man-1 ( Pichia pastoris Man-1).
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