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New (R)-transaminase from Trichoderma reesei and application thereof

A transaminase and amino acid technology, which is applied in the direction of transferase, enzyme, and the introduction of foreign genetic material using carriers, etc., can solve the problems of transaminase and other problems, and achieve good temperature stability and broad substrate spectrum.

Inactive Publication Date: 2015-05-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report of (R)-transaminase in China

Method used

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  • New (R)-transaminase from Trichoderma reesei and application thereof
  • New (R)-transaminase from Trichoderma reesei and application thereof
  • New (R)-transaminase from Trichoderma reesei and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 The preparation method of new (R)-transaminase HTR

[0029] (1) Construction of engineering bacteria: The coding gene (SEQ NO.1) of the new (R)-transaminase HTR was obtained by total gene synthesis (codon optimization was carried out according to the codon preference of Escherichia coli, and the N-terminal was added with 6×His-tag in order to purify the target protein (HTR) with a nickel affinity column), and connect to the pET32a expression vector Nde I / BamH I site. The recombinant plasmid is transferred into the host bacterial cell (preferably Escherichia coli BL21 (DE3)), and the corresponding engineering strain is obtained.

[0030] (2) Expression and purification: LB medium was used to culture in a shaker at 37°C until logarithmic phase, and after cooling to 25°C, IPTG with a final concentration of 0.1 mM was added to induce expression in a shaker at 25°C for 12 hours. Collect the bacteria by centrifugation at 4°C and 6,000 rpm, wash twice with sodium p...

Embodiment 2

[0032] Example 2 Enzymatic properties of new (R)-transaminase HTR

[0033] The optimum pH value, optimum reaction temperature and temperature stability of the new (R)-transaminase HTR were examined.

[0034] Simple reaction formula:

[0035] Activity assay reaction system: 1 ml of pH buffer, PLP (20 μM), (R)-1-phenylethylamine (10 mM), pyruvic acid (10 mM). The reaction was initiated by adding an appropriate amount of neo(R)-transaminase HTR, and terminated with perchloric acid after half an hour of reaction. After the reaction was terminated, the amount of acetophenone generated by the reaction was determined by HPLC to determine the size of the enzyme activity.

[0036] Determination of the optimum reaction pH value: adopt the bioassay reaction system, the reaction temperature is 30°C, measure at a series of different pH values ​​(pH=5, 6, 7, 7.4, 7.8, 8, 8.4, 8.8, 9, 9.4, 9.6 , 10) The enzymatic activity of the new (R)-transaminase HTR. The buffers used in different p...

Embodiment 3

[0040] Example 3 The substrate spectrum and application of the new (R)-transaminase HTR

[0041] (1) Amino donor substrate spectrum of the new (R)-transaminase HTR.

[0042] Simple reaction formula:

[0043] Reaction conditions: 1ml of reaction system. Add pyridoxal phosphate (20μM), amino donor substrate (10mM, racemate 20mM) and pyruvate (10mM) into sodium phosphate buffer (100mM, pH7.4), mix well and add appropriate amount of HTR enzyme solution Start the reaction, react in a 30°C water bath for 30 minutes, and then terminate the reaction with 375ul of 16% perchloric acid.

[0044] Calculation of relative activity: Calculate the relative activity of each substrate by detecting the decrease of pyruvate in the reaction system. The liquid phase detection conditions of pyruvate are: Aminex HPX-87H column (Bio-Rad, USA), column temperature 45 ° C, with 5 mM H 2 SO 4 As the mobile phase, the flow rate is 0.6ml / min, and the ultraviolet absorption value at 205nm is detected....

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Abstract

The invention discloses a new (R)-transaminase and application thereof. The (R)-transaminase derives from Trichoderma reesei QM6a (named HTR), its gene nucleotide sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No.2. The new (R)-transaminase HTR is a strict (R) stereoselective Omega-transaminase, has a broad substrate spectrum, and has great application potential in bio-production of chiral amines and unnatural amino acids.

Description

technical field [0001] The invention relates to the field of biocatalysis, and mainly relates to a new (R)-transaminase derived from Trichoderma reesei and its application in the production of chiral amines and unnatural amino acids. Background technique [0002] Chiral amines and unnatural amino acids have a wide range of applications in high value-added fields such as medicine, food, and cosmetics. For example (R)-4-phenylbutyl-2-amine (precursor of antihypertensive drug Dilevalol), phenylisoserine (β-amino acid, side chain moiety of anticancer drug paclitaxel) and cispentacin (cyclic β- Amino acids, antifungal drugs) are all key intermediates or key chiral modules for drug synthesis, and unnatural amino acids such as D-alanine are increasingly used in advanced cosmetics. In addition, studies have shown that peptides containing unnatural amino acids are more stable to protease degradation than natural peptides and still retain their biological activity. This shows that u...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/63C12P13/00
CPCC12N9/1096C12P13/04C12Y206/01
Inventor 朱敦明姜进举陈曦吴洽庆冯进辉马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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