Method for preparing rebuilding heat-resistant xylose/dextrose isomerase by employing pHsh expression system

Abstract

A preparing method of recombination heat resistance xylose / glucose isomerase, including: first, extracting genome DNA of thermozymes, amplificating PCR to obtain isomerase gene, inserting the gene in gene to express carrier pHsh, inverting coliform bacteria to obtain isomerase expression plasmid; second, mutagisming gene, changing usage frequency of codon and two-level structure of mRNA to obtain optimized isomerase expression plasmid; third, shifting the isomerase expression plasmid or optimized isomerase expression plasmid into coliform bacteria to obtain gene strains, and expressing xylose / glucose isomerase by thermally stimulating and inducing; finally, collecting cells, rupturing and centrifugating to obtain rough enzyme liquid, further purifying to obtain 90% purness of recombination heat resistance xylose / glucose isomerase. The invention first uses expression carrier pHsh to efficent express heat resistance xylose / glucose isomerase gene of thermozymes, producibility of recombination enzyme using this technque is more than 200 mg / L, the product has good stability and the method is easy to industrialized produce.

Description

1. Technical field [0001] The invention relates to the fields of molecular biology, enzymology, bioinformatics, genetic engineering and the like. In particular, it relates to a method for preparing recombinant heat-resistant xylose / glucose isomerase by using a pHsh expression system. 2. Background technology [0002] Fructose syrup is also called High-Fructose Corn Syrup (HFCS) or isomerized syrup, which is obtained by enzymatically saccharifying starch through the isomerization of xylose / glucose isomerase, and part of the glucose Isogenic fructose, a mixed sugar syrup composed of glucose and fructose. High fructose syrup is colorless and odorless, has good fluidity at room temperature, is easy to use, and has a wide range of sources. Its price is lower than that of sucrose and its sweetness is comparable to that of sucrose. It can partially or even completely replace sucrose in beverage production and food processing, so it is becoming more and more popular. By people's a...

AI Technical Summary

Problems solved by technology

However, due to the harsh growth conditions of thermophiles and the low natural expression of xylose / glucose isomerase, it is difficult to obtain a large amount of industrial enzymes from natural strains

At the same time, because the codon preference and the structure of the gene at room temperature in the thermophilic bacteria are quite different from those of the normal temperature bacteria, the expression level of the natural gene in the genetically engineered bacteria is usually low.

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