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L-pantoyl lactone dehydrogenase derived from Cnuibacter physcomitrellae

A technology of pantolactone and ester dehydrogenase, which is applied in the direction of enzymes, oxidoreductases, and microbial-based methods, can solve the problems of unknown coding genes and hinder applications, so as to reduce the use of acid and alkali, Simplify the reaction process and avoid cumulative effects

Active 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

In addition, although the L-pantolactone dehydrogenase derived from Nocardia stellatus has been studied in detail (Kataoka M, Shimizu S, Yamada H. Purification and characterization of a novel FMN-dependent enzyme :membrane-bound L-(+)-pantoyl lactone dehydrogenase from Nocardiaasteroides.European Journal of Biochemistry,1992,204,799-806), and its coding gene is still unknown, hindering its further application in biocatalysis
[0004] At present, there is no report of L-pantolactone dehydrogenase from Cnuibacter physcomitrellae, and there is no report on the use of L-pantolactone dehydrogenase from Cnuibacter physcomitrellae for multi-enzyme cascade catalysis of L-pantolysis A Report on the Synthesis of D-Pantolactone by Chiral Inversion of Acid Lactone

Method used

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  • L-pantoyl lactone dehydrogenase derived from Cnuibacter physcomitrellae
  • L-pantoyl lactone dehydrogenase derived from Cnuibacter physcomitrellae
  • L-pantoyl lactone dehydrogenase derived from Cnuibacter physcomitrellae

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1 Construction and expression of L-pantolactone dehydrogenase genetically engineered bacteria

[0073] Using the published oxidoreductase encoding gene from Cnuibacter physcomitrellae (GenBank accession number is ARJ05030.1), after codon optimization, L-pantoic acid was artificially synthesized (gene synthesis service provided by Suzhou Jinweizhi Biotechnology Co., Ltd.) The gene encoding lactone dehydrogenase has a nucleotide sequence as shown in SEQ ID NO:1.

[0074] The gene encoding L-pantolactone dehydrogenase (CphLPLDH) derived from Cnuibacter physcomitrellae was inserted into the HindIII and XhoI sites of plasmid pET-28b to obtain recombinant plasmid pET-28b-CphLPLDH. Transfer pET-28b-CphLPLDH into E.coli BL21(DE3) to obtain genetic engineering bacteria E.coli BL21(DE3) / pET-28b-CphLPLDH.

[0075] The genetically engineered bacteria E.coli BL21(DE3) / pET-28b-CphLPLDH were inoculated in LB liquid medium containing a final concentration of 100 μg / mL kanamyci...

Embodiment 2

[0076] The investigation of the substrate specificity of embodiment 2L-pantolactone dehydrogenase

[0077] The wet thallus obtained after the induced expression of genetically engineered bacteria E.coli BL21(DE3) / pET-28b-CphLPLDH was used as a biocatalyst. Using 100mM D-pantoate lactone, L-pantoate lactone, DL-pantoate lactone, D-pantoate and L-pantoate as substrates, the whole-cell catalysis was used to investigate the origin of Substrate specificity of the L-pantolactone dehydrogenase of Cnuibacter physcomitrellae. The reaction system for the dehydrogenation of L-pantolactone dehydrogenase catalyzed by L-pantolactone dehydrogenase is 5 mL, containing respectively: 1 g of wet bacteria, 100 mM substrate and 200 mM phosphate buffer (pH 7.0). The reaction solution was added to a three-necked flask, and the reaction conditions were maintained at 30°C, 600rpm and pH 7.0 under magnetic stirring, and the catalytic process was added dropwise with 1M Na 2 CO 3 The solution maintain...

Embodiment 3

[0081] Example 3D- Acquisition of Ketopantolactone Reductase Encoding Gene

[0082] Using the published reductase encoding gene (GenBank accession number: CAA98692.1) derived from Saccharomyces cerevisiae, after codon optimization, artificially synthesized (gene synthesis service provided by Suzhou Jinweizhi Biotechnology Co., Ltd.) D- The gene encoding ketopantolactone reductase has a nucleotide sequence as shown in SEQ ID NO:3.

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Abstract

The invention relates to the technical field of genetic engineering, particularly relates to L-pantoyl lactone dehydrogenase and application thereof, and discloses L-pantoyl lactone dehydrogenase derived from Cnuibacter physcomitrellae for the first time. The L-pantoyl lactone dehydrogenase has excellent enzyme activity on L-pantoyl lactone and can be applied to catalytic synthesis of D-pantoyl lactone. A system for chiral inversion synthesis of D-pantoyl lactone with L-pantoyl lactone by multi-enzyme cascade catalysis is constructed, and a genetically engineered bacterial cell co-expressing L-pantoyl lactone dehydrogenase, D-ketopantolactone reductase and glucose dehydrogenase is used as a catalyst, accumulation and spontaneous hydrolysis of the intermediate product ketopantolactone is avoided, and the steps of separating the intermediate product, racemizing the L-pantoyl lactone, lactonizing D-pantoic acid under an acidic condition are omitted, so that the reacting process is simplified, use of acid and alkali is reduced, and the reaction efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, more specifically, to L-pantolactone dehydrogenase derived from Cnuibacterphyscomitrellae, its encoding gene, vector, recombinant cell and application thereof. 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-pantoate lactone is an important raw material for the synthesis of calcium D-pantothenate. In industrial production, DL-pantoate lactone is first synthesized by chemical method, and then selectively hydrolyzed by lactone hydrolase D-pantoate lactone in D-pantoate lactone generates D-pantoate, and then separates D-pantoate and L-pantoate lactone, and the separated D-pantoate is acidified into a ring to form D-pantoate Lactone, and L-pantoacid lactone is reused after racemization. Therefore, although the chiral resolution metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/04C12R1/19
CPCC12N9/0006C12N15/70C12P17/04C12Y101/01168C12Y101/99027
Inventor 应向贤汪钊赵嫚程先锋林行殷杭华陈梁毛王伟张连春白彦兵余建新娄波金文究汪军刘一骏
Owner HANGZHOU XINFU TECH CO LTD
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