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Creatinase mutant with improved heat stability

A creatine hydrolase and mutant technology, applied in the field of enzyme engineering, can solve the problems of decreased enzyme activity and unsatisfactory thermal stability, and achieve the effect of improving thermal stability

Active Publication Date: 2016-08-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported optimal reaction temperature of creatine hydrolase is 30-40°C, but the thermal stability is not very ideal, and it is relatively stable below 45°C. Once the temperature is higher than 45°C, the enzyme activity will drop rapidly

Method used

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  • Creatinase mutant with improved heat stability
  • Creatinase mutant with improved heat stability
  • Creatinase mutant with improved heat stability

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The acquisition of embodiment 1 high thermostability mutant strain

[0014] Using the site-directed mutagenesis kit (TaKaRa), design multiple pairs of primers, and use the vector pET20J (highly efficient heterologous expression and application analysis of Arthrobacter tobacco creatinase [J]. Food and Biotechnology Journal) to pair the 368th and 368th primers Saturation mutations were performed at two sites at position 195. The nucleotide sequence encoding wild creatine hydrolase is shown in SEQ ID NO.6.

[0015] The PCR reaction conditions were 98°C for 3min, 34 cycles (98°C for 3min, 58°C for 30S, 72°C for 1min30S), and 72°C for 10min. PCR amplification system: template 1 μl, upstream and downstream primers 1 μl each, 2x PrimeStar 24 μl, sterilized double distilled water 24 μl. After PCR, add 5 μl of FD Buffer and 1 μl of DpnI to digest for 1 hour.

[0016] The plasmid containing the gene encoding the mutant was transformed into E.coli BL21, and the selected transfo...

Embodiment 2

[0017] The purification of embodiment 2 creatine hydrolase

[0018] After crushing Escherichia coli, carry out ammonium sulfate precipitation, select the precipitation with 55%--75% ammonium sulfate saturation, dissolve the protein precipitate with a small amount of phosphate buffer (pH 7.0), and dialyze for 24 hours to remove ammonium sulfate in the enzyme solution. According to the isoelectric point properties of creatine hydrolase, the QFF column was selected for ion exchange purification. The QFF column was equilibrated with phosphate buffer for 30 minutes, the crude enzyme solution was injected into the purification column, and the target protein was eluted with phosphate buffer containing 1M NaCl.

Embodiment 3

[0019] The property determination of the pure enzyme liquid of embodiment 3 mutants

[0020] Dilute each of the purified creatine hydrolase mutants, measure the enzyme activity of the mutants at 50°C for different times and calculate their half-life (t 1 / 2 , min), five mutants V368L, K195V, K195T, K195C, K195L were found to be 6.9, 3.7, 3.4, 1.6, 1.2 times higher than WT (as shown in Table 1). In addition, K195C's K m Compared with WT, the specific enzyme activity of K195V and K195C increased by 41.9% and 47.9%, respectively.

[0021] Table 1 Enzymatic Properties

[0022]

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Abstract

The invention discloses a creatinase mutant with improved heat stability, belonging to the field of enzyme engineering. A nucleotide sequence for coding the creatinase mutant is as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5. Compared with the half-life period of wild type creatinase, the half-life period of the creatinase mutant is respectively increased by 6.9, 3.7, 3.4, 1.6 and 1.2 times, so that the creatinase mutant is more suitable for industrial application.

Description

technical field [0001] The invention relates to a creatine hydrolase mutant with improved thermal stability, which belongs to the field of enzyme engineering. Background technique [0002] Creatinase (Creatinase, EC 3.5.3.3, referred to as CRE) is an essential enzyme for the detection of creatinine in enzymatic detection methods, mainly derived from microorganisms. In some studies, it was found that strains of some genera could induce the production of creatine hydrolase and accumulate in cells. These bacteria include Pseudomonas, Clostridium, Flavobacterium, Bacillus, Alcaligenes, and the like. However, because the creatine hydrolase yield of the original bacteria is very low, and the price of the inducer is high, it is not suitable for large-scale industrial production. At the same time, the analysis of the properties of creatine hydrolase found that it has the characteristics of low substrate affinity and poor thermal stability. However, in practical applications, a la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/70C12Q1/34
CPCC12N9/78C12Q1/34C12Y305/03003G01N2333/986
Inventor 刘松李江华阮洁陈坚堵国成
Owner JIANGNAN UNIV
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