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Connecting peptide, fusion protein containing connecting peptide and application thereof

A technology of fusion protein and secondary alcohol, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of reduced catalytic specificity constant and low efficiency of coenzyme cycle, and achieve the effects of high-efficiency preparation, improved catalytic properties, and reduced costs

Active Publication Date: 2022-03-22
CHINA NAT INST OF STANDARDIZATION
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the problems that the existing fusion enzyme coenzyme regeneration system is limited to the regeneration of reduced coenzyme, the catalytic specificity constant decreases after enzyme fusion, and the efficiency of coenzyme cycle is not high, the present invention provides a connecting peptide and a fusion protein containing the connecting peptide and its application, the fusion protein is a fusion protein involving alcohol dehydrogenase and NAD(P)H oxidase, the fusion protein is aimed at the regeneration of oxidized coenzymes, and can improve the interaction between alcohol dehydrogenase and NAD(P)H oxidase activity, improve its catalytic properties, and reasonably control the distance between the two enzymes by adjusting the length of the connecting peptide, realize the spatial proximity effect of the protein in vitro, and improve the recycling efficiency of the coenzyme

Method used

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  • Connecting peptide, fusion protein containing connecting peptide and application thereof
  • Connecting peptide, fusion protein containing connecting peptide and application thereof
  • Connecting peptide, fusion protein containing connecting peptide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Embodiment 1 Fusion protein construction of alcohol dehydrogenase and NAD(P)H oxidase

[0079] The alcohol dehydrogenase and NAD(P)H oxidase involved in this example are all derived from the hyperthermophilic bacteria Thermococcuskodakarensis KOD1, named TkADH and TkNOX respectively, and the accession numbers in Genebank are BAD85034.1 and BAD84493.1 respectively .

[0080] For the heterologous expression and purification of TkADH and TkNOX, please refer to the literature (Wu Xi, et al.Thermostablealcohol dehydrogenase from Thermococcus kodakarensis KOD1 for enantioselectivebioconversion of aromatic secondary alcohols. Applied and Environmental Microbiology, 2013, 79:2209-2217.) and (Wu Xi, et al. al.Application of a novelthermostable NAD(P)H oxidase from hyperthermophilic archaeon for regeneration of both NAD + and NADP + . Biotechnology and Bioengineering, 2012, 109:53-62.).

[0081] The construction of the fusion protein involved in the present invention is throug...

Embodiment 2

[0108] Activity analysis of embodiment 2 fusion protein

[0109] By the method in Example 1, construct and express fusion protein ADH-R0-NOX, ADH-R1-NOX, ADH-R2-NOX, ADH-R3-NOX, NOX-R0-ADH, NOX-R1-ADH, NOX -R2-ADH and NOX-R3-ADH, the fusion protein was purified by heat treatment. In order to investigate the intrinsic changes in the activities of the eight fusion proteins with different linking peptide lengths and fusion directions, the alcohol dehydrogenase activity and NAD(P)H oxidase activity were measured at 70°C.

[0110] (1) Determination of Alcohol Dehydrogenase Activity

[0111] The method for detecting the activity of alcohol dehydrogenase (ADH) in the fusion protein is as follows: the reaction system is 750 μL, including 50 mM glycine-sodium hydroxide buffer solution (pH 9.0), 100 mM (RS)-1-phenylethyl alcohol, 1 mM NAD + , add an appropriate amount of enzyme solution (when measuring the activity of the fusion protein, the enzyme solution is the fusion protein; when...

Embodiment 3

[0122] The kinetic parameter determination of embodiment 3 fusion protein

[0123] In order to minimize the error caused by the volatilization of the substrate at high temperature, and to better compare the difference in coenzyme regeneration efficiency between the fusion protein and its equal-activity single-enzyme mixed system, the reaction temperature was set at 40°C. Firstly, the fusion protein was tested at 40°C for the coenzyme NAD + And the kinetic parameters of NADH. The buffer used in the determination of alcohol dehydrogenase and NAD(P)H oxidase activity is the buffer to be used in the coenzyme regeneration reaction—50mM Gly-NaOH buffer (pH 9.0).

[0124] The specific method is as follows: For the ADH part, add 100 mM (RS)-1-phenylethanol to the buffer, and change the NAD in the range of 0-1.0 mM + Concentration of NAD + Initial rate of reduction; for the NOX fraction, the initial rate of NADH oxidation was determined by varying the concentration of NADH in the ra...

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Abstract

The invention discloses a connecting peptide, a fusion protein containing the connecting peptide and applications thereof. The connecting peptide is R0, R1, R2 or R3, wherein the amino acid sequence of R0 is AAA, the amino acid sequence of R1 is EAAAK, and the amino acid sequence of R2 is (EAAAK) 2 , the amino acid sequence of R3 is (EAAAK) 3 . The connecting peptide designed in the present invention can be used for molecular transformation of fusion protein. Through the design of the connecting peptide of the fusion protein of alcohol dehydrogenase and NAD(P)H oxidase, the activity of alcohol dehydrogenase and NAD(P)H oxidase is improved, the catalytic properties are improved, and the length of the connecting peptide is adjusted. The distance between the two enzymes is reasonably controlled to realize the spatial proximity effect in vitro, improve the circulation efficiency of the coenzyme, and realize the efficient regeneration of the oxidized coenzyme. Further use of a single fusion protein can efficiently prepare optically pure chiral alcohols, simplify the system, reduce costs, and provide a new tool for the production of intermediates in the pharmaceutical and chemical industries.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to a connecting peptide, a fusion protein containing the connecting peptide and applications thereof. Background technique [0002] Chiral alcohols are important intermediates in the pharmaceutical and chemical industries. The use of alcohol dehydrogenase (ADH, alcoholehydrogenase) for the production of chiral alcohols has a series of advantages over chemical methods: the reaction has a high degree of chemical, stereo and regioselectivity, the catalytic conditions are mild and the catalytic process is environmentally friendly. Alcohol dehydrogenase needs to continuously consume nicotinamide coenzyme NAD or NADP in the process of catalyzing the interconversion of alcohol and aldehyde / ketone. However, coenzyme is expensive and unstable. In order to reduce the cost of industrial application of alcohol dehydrogenase, it is necessary to develop an eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/08C07K7/06C07K7/08C12N15/11C12N9/20C12N9/04C12N15/62C12P19/36C12P41/00C12P7/22C12P7/18
CPCC07K5/08C07K7/06C07K7/08C12N9/0006C12N9/0036C12P19/36C12P41/001C12P7/22C12P7/18C12Y101/01001C12Y106/03001C07K2319/00Y02P20/584
Inventor 吴希张翀云振宇赵琳吴琦
Owner CHINA NAT INST OF STANDARDIZATION
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