阅读说明：本技术 一种热稳定性提高的肌酸水解酶突变体 (Creatine hydrolase mutant with improved thermal stability ) 是由 刘松 李江华 阮洁 陈坚 堵国成 于 2016-05-04 设计创作，主要内容包括：本发明公开了一种热稳定性提高的肌酸水解酶突变体,属于酶工程领域。编码所述肌酸水解酶突变体的核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4或SEQ ID NO.5所示。所得肌酸水解酶突变体的半衰期相比野生型分别提高6.9、3.7、3.4、1.6、1.2倍,更适合工业应用。(The invention discloses a myohydrolase mutant with improved thermal stability, and belongs to the field of enzyme engineering. The nucleotide sequence for coding the creatine hydrolase mutant is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5. Compared with the wild type, the half-life period of the creatine hydrolase mutant is respectively improved by 6.9, 3.7, 3.4, 1.6 and 1.2 times, and the creatine hydrolase mutant is more suitable for industrial application.)

1. The creatine hydrolase is characterized in that a nucleotide sequence for coding the creatine hydrolase is shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 5.

2. A nucleotide sequence encoding the creatine hydrolase of claim 1.

3. A vector or cell comprising the nucleotide sequence of claim 2.

4. A method for obtaining the creatine hydrolase of claim 1, comprising the steps of: PCR is carried out to obtain nucleotide sequence for coding mutant, expression vector is connected, E.coli BL21 is transformed, and recombinant bacteria are cultured by TB culture medium to express mutant.

5. A genetically engineered bacterium for expressing the creatine hydrolase of claim 1, wherein pET-20b is used as a vector to construct a recombinant expression vector, and a host BL21(DE3) is transformed.

6. Use of the creatine hydrolase of claim 1 for detecting creatinine.

7. A creatinine assay kit comprising the creatine hydrolase according to claim 1.

8. The use of the creatinine assay kit of claim 7 for the detection of creatinine.

Technical Field

The invention relates to a myohydrolase mutant with improved thermal stability, belonging to the field of enzyme engineering.

Background

Creatine hydrolase (EC 3.5.3.3, CRE) is an essential enzyme for detecting creatinine in an enzymatic assay, and is mainly derived from microorganisms. In some studies it was found that strains of some genera are capable of inducing the production of creatine hydrolase and accumulate in the cells. Such bacteria include Pseudomonas, Clostridium, Flavobacterium, Bacillus, Alcaligenes, and the like. However, the yield of creatine hydrolase from the original strain is very low, and the price of the inducer is high, so that the creatine hydrolase is not suitable for industrial large-scale production. Meanwhile, the creatine hydrolase is characterized by low substrate affinity, poor thermal stability and the like through property analysis. In practical application, a large amount of enzyme is required, which is not favorable for industrial production.

The strains which are deeply analyzed for the nature of creatine hydrolase at present are mainly pseudomonas putida, arthrobacter and alcaligenes. The creatine hydrolysis enzyme properties of different sources are comprehensively analyzed, the optimum reaction pH range of most creatine hydrolysis enzymes is 7.0-8.0, and the creatine hydrolysis enzymes are stable under the conditions of neutrality, weak base and weak acid. The reported optimum reaction temperature of the creatine hydrolase is 30-40 ℃, the thermal stability is not ideal, the creatine hydrolase is relatively stable below 45 ℃, and once the temperature is higher than 45 ℃, the enzyme activity can be rapidly reduced. Therefore, there is a need for a thermal stability study of creatine hydrolase.

Disclosure of Invention

The invention aims to solve the problem of providing a creatine hydrolase mutant with improved thermal stability.

The nucleotide sequence for coding the creatine hydrolase mutant is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

The invention also provides a method for obtaining the creatine hydrolase mutant, which comprises the following steps: PCR is carried out to obtain nucleotide sequence for coding mutant, expression vector is connected, E.coli BL21 is transformed, and recombinant bacteria are cultured by TB culture medium to express mutant.

The invention also provides a genetic engineering bacterium for expressing the creatine hydrolase mutant, which is a recombinant expression vector constructed by taking pET-20b as a vector and transforming a host BL21(DE 3).

The invention also provides a creatinine detection kit containing the creatine hydrolase mutant.

The invention has the beneficial effects that: the thermal stability of the creatine hydrolase is improved, and the half-life period of the obtained creatine hydrolase mutant is respectively improved by 6.9, 3.7, 3.4, 1.6 and 1.2 times compared with that of the wild type, so that the creatine hydrolase mutant is more suitable for industrial application.

Detailed Description

LB culture medium: 10g/L of tryptone and 5g/L, NaCl 10g/L of yeast powder.

TB culture medium: peptone 12g/L, yeast powder 24g/L, glycerin 10g/L, KH₂PO₄ 2.32g/L、K₂HPO₄16.43g/L。

The enzyme activity of the creatine hydrolase is determined by adopting a spectrophotometric method. Definition of unit enzyme activity: the amount of enzyme required to hydrolyze creatine to 1. mu. mol urea in 1 min. The enzyme activity determination conditions are as follows: mu.L of creatine solution was added to the tube and allowed to equilibrate for 5min at room temperature. Then 100 mul of enzyme solution to be tested is added, the reaction is carried out for 10min at 37 ℃, then 2mL of p-dimethylamine benzaldehyde solution is added into the reaction system to terminate the reaction, the reaction system is placed at 25 ℃ for incubation for 20min, the absorbance value is measured at 435nm, and the zero adjustment is carried out by water. The blank tube is that p-dimethylaminobenzaldehyde is added into creatine solution firstly, then enzyme solution to be tested is added, and other steps are consistent with those of the measuring tube.