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Ketopantolactone reductase and application thereof

A pantolactone and reductase technology, applied in oxidoreductase, application, genetic engineering and other directions, to achieve the effect of excellent enzyme activity, avoid additional addition, and optimize the reaction system

Inactive Publication Date: 2019-11-01
HANGZHOU XINFU TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report that the aldehyde and ketone reductase from fission yeast has D-ketopantolactone reductase activity, and there is no report that the aldehyde and ketone reductase from fission yeast is used for D-pantolactone Reports on Asymmetric Synthesis

Method used

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  • Ketopantolactone reductase and application thereof
  • Ketopantolactone reductase and application thereof
  • Ketopantolactone reductase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Construction and Expression of Fission Saccharomyces Ketopantolactone Reductase Genetic Engineering Bacteria

[0066] The fission yeast ketopantolactone reductase gene KPR uses the genomic DNA of fission yeast (Schizosaccharomycespombe, from the China Industrial Microbiology Culture Collection Management Center, strain number: CICC 1056) as a template, and uses the designed primer F1( The nucleotide sequence is shown in SEQ ID NO: 6) and R1 (the nucleotide sequence is shown in SEQ ID NO: 7) for PCR amplification, and the amplification system is shown in Table 1. The PCR reaction process is as follows: 95°C pre-denaturation for 2 minutes; then, denaturation at 95°C for 20 sec, 55°C for 20 sec, 72°C for 20 sec as a cycle, and repeat this cycle 35 times; finally, 72°C for 5 min. PCR products were detected and recovered by 1% agarose gel electrophoresis. The purified PCR product was connected with the vector pEASY-Blunt E1, and the connected product was transform...

Embodiment 2

[0070] Example 2 Separation and Purification of Fission Yeast Ketopantolactone Reductase

[0071] Add appropriate amount of Tris-HCl (pH 8.0) damping solution to the wet thalline collected in Example 1 by the ratio of 1g wet thalline plus 15mL 50mM Tris-HCl buffer (pH 8.0), and ultrasonically break it for 20min at 500W (working 2s, 6s interval), the crushed solution was centrifuged at 4°C and 10000rpm for 10min, and the centrifugation was repeated three times to obtain the supernatant crude enzyme solution.

Embodiment 3

[0074] Example 3 Determination of Enzyme Activity of Fission Yeast Ketopantolactone Reductase

[0075] Enzyme activity assay method: Standard enzyme activity assay system (2.5mL) contains: 0.15mM NADPH, 0.6μg / mL fission yeast ketopantolide reductase, 10mM ketopantolide (ketopantolide 0.2MKH pH 2.0 for hydrochloride 2 PO 4 -HCl solution is prepared in the form of 1M substrate solution and added, the amount of substrate solution added is based on the amount of ketopantoolactone, and the final concentration of ketopantoolactone in the reaction system is 10mM), with 0.2M 2 HPO 4 -NaH 2 PO 4 Buffer (pH 6.0) was the reaction medium. The stated concentrations all refer to the final concentrations in the assay system. The reaction was carried out at 55°C, NADPH and the substrate were added last, and the enzyme activity was determined by detecting the change of the absorbance value of the reaction system at 340nm per minute (the molar coefficient ε of NADPH 340 =6.22mM -1 cm -...

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Abstract

The invention relates to the technical field of microbial gene engineering, in particular to brand-new ketopantolactone reductase derived from schizosaccharomyces pombe and application of the brand-new ketopantolactone reductase. The ketopantoate reductase has excellent enzymatic activity to ketopantolactone and can catalyze asymmetric reduction of the ketopantolactone to generate D-pantolactone.A one-bacterium double-enzyme reaction system is further constructed, the ketopantolactone reductase generated by double-enzyme recombinant cell induction is coupled with glucose dehydrogenase, asymmetric reduction of a substrate, namely the ketopantolactone, is catalyzed to generate the D-pantolactone, beta-nicotinamide-adenine dinucleotide phosphate (NADP<+>) is reduced into nicotinamide adeninedinucleotide phosphate (NADPH) through dehydrogenation of a cosubstrate, namely glucose, and thus coenzyme circulation is realized. The catalytic efficiency of the glucose dehydrogenase is much higher than that of the ketopantolactone reductase, thus reduction reaction can be promoted to be conducted at the high speed, the reaction efficiency is greatly improved, and the cost is lowered.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a ketopantolactone reductase derived from Schizosaccharomyces pombe and its application. Background technique [0002] Calcium D-pantothenate, also known as vitamin B5, is a component of coenzyme A and has been widely used in industries such as medicine, food, feed and cosmetics. D-pantothenate lactone is an important raw material for the synthesis of D-calcium pantothenate. In industrial production, DL-pantoate lactone is first synthesized by chemical method, and then the D-pantoate lactone in the cyclic pantoate lactone is selectively hydrolyzed by lactone hydrolase to generate D-pantoate. Then separate D-pantoate and L-pantoate lactone, the separated D-pantoate is acidified into a ring to form D-pantoate lactone, and L-pantoate lactone after racemization Reuse. Therefore, although the chiral resolution method catalyzed by hydrolase is a mature process,...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12P17/04
CPCC12N9/0006C12N15/70C12P17/04
Inventor 应向贤汪钊赵嫚程先锋林行白彦兵张丽陈梁殷杭华张连春毛王伟余建新王春霞郑世明何敏
Owner HANGZHOU XINFU TECH CO LTD
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