A kind of alginate lyase alg7d and its preparation method and application
A technology of alginate lyase and gene, which is applied in the field of high-efficiency preparation of alginate lyase, can solve the problems of increased difficulty, easy loss, and unstable expression of free plasmid vectors, and achieves difficult loss, stable expression, and high-efficiency secretion expression Effect
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Embodiment 1
[0023] Example 1 Codon optimization and total gene synthesis of alginate lyase gene
[0024] Under the premise of not changing the amino acid sequence, the codons of the alginate lyase coding gene derived from Saccharophagus degradans 2-40 (Saccharophagusdegradans 2-40) were optimized, and all the optimized codons were Pichia pastoris preferred codons The specific sequence is shown in SEQ ID NO.2. The identity (identity) of the optimized gene sequence and the original sequence of alginate lyase Alg7D (as shown in sequence SEQ ID NO.3, GenBank accession number: CP000282.1) is 75%. It should be noted that, in order to enable greater expression of alginate lyase in Pichia pastoris, only the gene encoding the carboxy-terminal catalytic region (amino acids 331-611) of the original alginate lyase was selected for optimization and expression, and the The amino-terminal glycan-binding domain. The optimized gene sequence was entrusted to Beijing Qingke Xingye Biotechnology Co., Ltd. ...
Embodiment 2
[0025] Example 2 Construction of expression vector of alginate lyase gene alg7D
[0026] Firstly, the cloning vector containing the alginate lyase gene alg7D was double-digested with restriction endonucleases Xho I and Not I to obtain the target gene fragment, and the expression vector pGBG1 was double-digested with the same endonuclease, and recovered large fragments. The two recovered products were connected to obtain a recombinant vector named alg7D-pGBG1. In order to confirm that the target alginate lyase gene has been constructed into the vector, the recombinant vector was double digested with Xho I and Not I, and the product was subjected to agarose gel electrophoresis. The results are as follows: figure 1 shown. according to figure 1 It can be seen that after double enzyme digestion, the target gene fragment appeared between 750bp and 1000bp, which was consistent with the fragment length of alg7D of 879bp.
Embodiment 3
[0027] Example 3 Screening of alginate lyase Pichia pastoris engineering bacteria and preparation of alginate lyase
[0028] After the obtained recombinant plasmid alg7D-pGBG1 was linearized by the restriction endonuclease BglII, it was separated by gel electrophoresis and a large nucleic acid fragment containing the target gene was excised, and introduced into Pichia pastoris GS115 by electroporation, and passed through the histidine auxotrophic MD plate. Recombinant colonies were obtained by screening. Eight of the colonies were picked and streaked onto a BMMY agar plate containing 0.2% sodium alginate, cultured at 30°C for 48 hours, then poured into 10% cetylpyridinium chloride (Cetylpyridinium chloride, CPC) aqueous solution for color development, and found Only clone No. 5 exhibited alginate-cleaving activity, see figure 2 , is the primary screening chart for the enzymatic activity of the recombinant expression Pichia pastoris GS115 strain sodium alginate substrate plat...
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