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A kind of improved highly active thermostable creatine hydrolase and its application

A technology of creatine hydrolase and amino acid, applied in the direction of hydrolase, application, enzyme, etc., can solve the problem of thermal stability not suitable for industrial production, and achieve the effect of improving thermal stability and specific activity

Active Publication Date: 2020-07-14
湖南艾科瑞生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme is also not suitable for the needs of industrial production in terms of thermostability and enzyme activity.

Method used

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  • A kind of improved highly active thermostable creatine hydrolase and its application
  • A kind of improved highly active thermostable creatine hydrolase and its application
  • A kind of improved highly active thermostable creatine hydrolase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Obtaining of highly active mutant sequences

[0032] Based on the sequence of SEQ ID NO: 1 (from the patent CN104109658B), a mutation library was established by error-prone PCR, and the mutated target protein SEQ ID NO was obtained through gene screening and enzyme expression thermal stability and enzyme activity screening : Optimized gene for 2T-creatinase and SEQ ID NO:3. SEQ ID NO: 1: creatinase nucleotide sequence

[0033]

[0034]

[0035]

[0036] SEQ ID NO: 2T-creatinase amino acid sequence

[0037]

[0038] SEQ ID NO: 3 T-creatinase nucleotide sequence

[0039]

Embodiment 2

[0040] Embodiment 2: Construction of the expression system of mutant creatine hydrolase gene

[0041]The expression vector was selected as pET20b(+), the upstream restriction site was selected as NdeI, and the downstream restriction site was XhoI according to the carrier. The obtained cloning vector pMD19-T containing the T-creatinase of SEQ ID NO: 3 and the expression vector pET20b(+) were subjected to double enzyme digestion respectively. After digestion at 37° C. for 60 min, gel electrophoresis verified and the digestion products were recovered. The recovered expression vector was connected with the target gene, and the connection solution was transformed into the expression host E.coli-BL21, and the recombinant clone was verified to be correct by PCR.

Embodiment 3

[0042] Example 3: Heterologous expression of T-creatinase in recombinant Escherichia coli

[0043] Glycerol tube: the concentration of glycerol is 20%; the composition of the seed medium is (g / L): peptone 10, yeast extract 5, NaCl10, pH7.0, 100 μg / mL ampicillin; the composition of the fermentation medium is (g / L) : Peptone 12, yeast extract 24, glycerin 4, KH 2 PO 4 2.31,K 2 HPO 4 16.43, 100 μg / mL ampicillin. Seed culture: transfer 0.4% inoculum amount from glycerol tubes into 20mL / 250mL Erlenmeyer flasks, rotate on a rotary shaker at 200rpm, cultivate at 37°C for 12 hours. Fermentation culture: Inoculate the cultivated seed culture solution into a 50mL / 500mL Erlenmeyer flask at an inoculation amount of 1% (v / v) for cultivation, the cultivation temperature is 37°C, and the shaker speed is 200rpm. When OD600=0.6, add IPTG with a final concentration of 0.6mmol / L to induce expression, cultivate for 10h, the creatine hydrolase production can reach 125.6U / mL, and the control u...

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Abstract

The invention relates to a mutant of creatinase. The specific activity and the heat stability of the creatinase are improved. Compared with wild-type creatinase, the mutant is more suitable for being applied to industrialization and has excellent market application prospects and economic values.

Description

technical field [0001] The invention relates to the field of biological fermentation, in particular to an improved high-activity heat-resistant creatine hydrolase and application thereof. Background technique [0002] Creatine hydrolase (creatinase or creatine amidohydrolase, EC 3.5.3.3, referred to as CRE) can catalyze the hydrolysis of creatine to produce urea and sarcosine. Creatine hydrolase has been found in many kinds of bacteria. Creatinine is the final product of phosphocreatine metabolism in the human body. It can be filtered by the kidneys, enter the urine from the blood, and be excreted from the body. The normal range of creatinine content in serum is between 35-150 μM, but when there is a problem with kidney function or muscle function, the content of creatinine will rise to 1000 μM. Therefore, creatinine levels in blood and urine can reflect the excretory function of the kidneys. Compared with chemical detection methods, enzymatic detection has more advantage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/78C12N15/70C12Y305/03003
Inventor 雷洁戴俊
Owner 湖南艾科瑞生物工程有限公司
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