Recombination expression of HPLC12 type ice structuring protein in bacillus subtilis and preparation method

Abstract

The invention discloses a bacillus subtilis recombinant strain which is obtained by introducing a HPLC12 type ice structuring protein gene into bacillus subtilis. The amino acid sequence of the HPLC12 type ice structuring protein is as shown in a sequence 2. The invention also provides a preparation method of the HPLC12 type ice structuring protein. The HPLC12 type ice structuring protein is obtained by fermenting any one of the bacillus subtilis recombinant strains in the claims 1-4. The preparation method has the beneficial effects that the bacillus subtilis has biological safety and good genetic stability and can persistently secrete expression foreign genes, a HPLC12 type ice structuring protein gene subjected to codon optimization is inserted behind a multiple-cloning site starting from a bacillus subtilis plasmid pAl12 so that the bacillus subtilis recombinant engineering strain which can stably and largely secrete the HPLC12 type ice structuring proteins can be obtained. The preparation method plays an important role in the biological preparation of antifreeze protein expression.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a recombinant expression and preparation method of HPLC12 ice structural protein in Bacillus subtilis. Background technique [0002] Ice structuring proteins (ISPs), also known as antifreeze proteins (AFPs), refer to a class of polypeptides produced by certain vertebrates, plants, fungi and bacteria. These peptides enable these species to survive in sub-zero temperatures. ISPs were originally discovered in the serum of marine fish in the polar regions of the earth. It can combine with ice crystals to prevent the formation and growth of ice nuclei in fish body fluids, thereby playing an antifreeze role. The antifreeze performance of ISPs is different from that of automobile antifreeze, and the freezing point depression is not proportional to the concentration. They do not function according to the laws of colligative numbers. In this way, they can be made to function as antifreez...

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Problems solved by technology

[0003]At present, there are three types of production methods for HPLC-type ice structural protein, one is obtained through chemical extraction and biological expression, but the chemical method has high energy input and produces a large amount of Chemical waste (toluene, benzene chloroform and ethyl acetate, etc.), and more than ten steps are required, the product separation and purification is difficult, and the cost is high. Although people have done a lot of research on chemical synthesis methods, they still cannot effectively solve the above problems. Seriously hinder the industrial production process of antifreeze protein; the second is to extract directly from the organism, which is extremely expensive and the yield is not high; the third is to use Saccharomyces cerevisiae and Escherichia coli for expression. With the rapid development of bioengineering technology, people try to change the production capacity of strains through genetic engineering, so that the fermentation unit is greatly improved. The yeast ferm

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