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Creatinase mutant with increased thermal stability

A technology of creatine hydrolase and thermal stability, which is applied in the direction of hydrolytic enzymes, enzymes, and microbial-based methods, and can solve problems such as unsatisfactory thermal stability and decreased enzyme activity

Active Publication Date: 2016-01-27
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 increased thermal stability
  • Creatinase mutant with increased thermal stability
  • Creatinase mutant with increased thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The acquisition of embodiment 1 high thermostability mutant strain

[0023] Using the site-directed mutagenesis kit (TaKaRa), design a pair of primers (as shown in Table 1), and use the constructed pET20J as a template to perform PCR to mutate the 358th valine inside the creatine hydrolase molecule to methionine , named V368M, 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, 2xPrimeStar 24 μl, sterilized double distilled water 24 μl. After PCR, add 5 μl of FDbuffer and 1 μl of DpnI to digest for 1 hour. The digested PCR product was purified, recovered and transformed using a gel recovery kit. Transformants were sequenced by Shanghai Sangon. The correctly sequenced transformant was named Pet20J-V368M.

[0024] Table 1

[0025]

Embodiment 2

[0026] Example 2 Verification of High Thermostable Creatine Hydrolase Mutant Strain

[0027] The plasmid with correct sequencing was transformed into E.coliBL21, and the transformants were selected and inoculated into LB liquid medium, cultured at 37°C for 12 hours, and transformed into TB medium with an inoculum size of 3%. When the bacteria grew to OD600 of 3, IPTG was added to make the final concentration 0.6mmol / L, and the culture temperature was lowered to 30°C for 8h. Collect the fermented bacteria. After ultrasonic crushing, the enzyme activity was measured at the same temperature for different time.

Embodiment 3

[0028] The purification of embodiment 3 creatine hydrolase

[0029] 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 (pH7.0), 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 min, the crude enzyme solution was injected into the purification column, and the target protein was eluted with phosphate buffer containing 1M NaCl.

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Abstract

The invention discloses a creatinase mutant with increased thermal stability, and belongs to the field of enzyme engineering. The mutant is obtained by mutating a 368th amino acid of a creatinase amino acid sequence. Creatinase roots in escherichia coli. The mutant is a mutant V368M obtained by mutating a 368th valine into methionine. An amino acid in creatinase protein rooting in the escherichia coli is subjected to site-specific mutagenesis, so that thermal stability of the creatinase is improved. Compared with the existing mutant V368M, the creatinase mutant with increased thermal stability has the advantages that half-life is prolonged for 5 times, and Tm value is increased by 3 DEG C.

Description

technical field [0001] The invention discloses a creatine hydrolase (Creatinase, EC3.5.3.3, CRE for short) mutant with improved thermostability, which belongs to the field of enzyme engineering. Background technique [0002] Creatine hydrolase is an essential enzyme for the detection of creatinine in enzymatic detection methods, and its main source is 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. Just need a large amount of enzymes in...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N1/21C12R1/19
CPCC12N9/78C12Y305/03003
Inventor 陈坚刘松阮洁冯岳李江华陈双全邱芳芳王晨蕾
Owner JIANGNAN UNIV
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