A kind of engineering bacteria expressing pectate endohydrolase and its application
A technology of hydrolase and pectic acid, applied in the fields of bioengineering, microbial engineering, and food engineering, can solve the problems of inhibition of pectinase expression, poor expression effect, and products easily mixed with endotoxin.
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Embodiment 1
[0028] Embodiment 1 npg gene design and npg / PMD18T plasmid construction
[0029] Using the npg gene derived from Aspergillus oryzae pectate endohydrolase A, 35 codons were optimized to make it suitable for the expression system of Bacillus subtilis, and its upstream and downstream were fused to encode specific polypeptides GSHS and SHS respectively The nucleotide sequence of (SSSSHHHHHHHHSSS and SSHHHHSS) was added with NdeI site sequence at the 5' end, and SalI site sequence was added at the 3' end, and the nucleotide sequence shown in SEQ ID NO.1 was chemically synthesized, and its amino acid sequence was as follows: Shown in SEQ ID NO.2.
[0030] Ligate the synthesized fragment with the PMD18T vector, transform DH5α (for specific transformation steps, refer to the manual of full gold DH5α), coat the transformed product on an LB plate containing 100 μg / ml ampicillin, and culture at 37°C overnight (10h-14h) to obtain a single clone For transformants, single clones were picke...
Embodiment 2
[0034] Embodiment 2 npg / pBSCWZ expression vector structure design and construction
[0035] The nucleotide sequence of the designed expression plasmid (npg / pBSCWZ) is shown in SEQ ID NO.3, and the plasmid structure is shown in figure 1 shown.
[0036] The vector used was the pBSCWZ vector constructed by the inventor (the nucleotide sequence of the vector is shown in SEQ ID NO.9), and the pBSCWZ plasmid and npg / PMD18T were digested with NdeI and SalI ( figure 2), the enzyme-digested product was then gel-recovered (see the OMEGA Gel Recovery Kit for details), and then ligated with T4 ligase to obtain the ligated product. The ligated product was transformed into E.coliDH5α competent cells, and cultured at 37°C overnight (10h ~14h) until a single clone grows, pick a single clone and put it in LB liquid medium containing 100μg / ml ampicillin, culture it at 37°C and 200rpm for 8h to obtain a bacterial liquid, and carry out colony PCR identification on the bacterial liquid, and the ...
Embodiment 3
[0037] Example 3 Construction of npg / pBSCWZ / B.subtilis 800 expression engineering bacteria
[0038] Transform the plasmid npg / pBSCWZ into B. subtilis 800, spread the transformation solution on an LB plate containing 100 μg / ml kanamycin, culture overnight at 37°C (10h-14h) until a single clone grows, and pick a single clone to The bacteria were cultured in LB liquid medium containing 100 μg / ml ampicillin at 37° C. and 200 rpm for 8 hours to obtain a bacterial solution. The bacterial solution was identified by colony PCR and sequenced. The primers used for colony PCR were F1 and R1 in Example 1. The monoclonal transformant npg / pBSCWZ / B.subtilis 800 (that is, the GSHS-NPG-SHS engineering strain) was verified. Transformation steps: refer to "Molecular Cloning Experiment Guide" (translated by Huang Peitang et al., published by Science Press in September 2002).
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