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L-pantolactone dehydrogenase derived from Nocardia cyriacigeorgica and application thereof

A technology of pantolactone and ester dehydrogenase, applied in application, enzyme, oxidoreductase, etc., can solve problems such as hindering application and unknown coding gene, so as to reduce the use of acid and alkali and simplify the reaction process , the effect of excellent enzyme activity

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] No L-pantolactone dehydrogenase from Nocardia cyriacigeorgica has been reported so far, and no L-pantolactone dehydrogenase from Nocardia cyriacigeorgica has been used to catalyze L-pantolysis in a multi-enzyme cascade A Report on the Synthesis of D-Pantolactone by Chiral Inversion of Acid Lactone

Method used

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  • L-pantolactone dehydrogenase derived from Nocardia cyriacigeorgica and application thereof
  • L-pantolactone dehydrogenase derived from Nocardia cyriacigeorgica and application thereof
  • L-pantolactone dehydrogenase derived from Nocardia cyriacigeorgica and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0073] Using the published oxidoreductase coding gene (GenBank accession number: CCF64149.1) from Nocardia cyriacigeorgica, 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 (NcyLPLDH) derived from Nocardia cyriacigeorgica was inserted into the Hind III and Xho I sites of plasmid pET-28b to obtain recombinant plasmid pET-28b-NcyLPLDH. Transfer pET-28b-NcyLPLDH into E.coli BL21(DE3) to obtain genetically engineered bacteria E.coli BL21(DE3) / pET-28b-NcyLPLDH.

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

Embodiment 2

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

[0077] The wet bacterium obtained after the induced expression of genetically engineered bacteria E.coli BL21(DE3) / pET-28b-NcyLPLDH 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 Nocardia cyriacigeorgica. 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

[0080] Example 3D- Acquisition of Ketopantolactone Reductase Encoding Gene

[0081] 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, in particular to L-pantolactone dehydrogenase and application thereof. The L-pantolactone dehydrogenase derived from Nocardia cyriacigeorgica is disclosed for the first time, the L-pantolactone dehydrogenase has good enzymatic activity for L-pantolactone and can be applied to the catalytic synthesis of D-pantolactone. A system for synthesizing D-pantolactone through multi-enzyme cascade catalyzed L-pantolactone chiral inversion is constructed, genetically engineered bacterial cells which co-express L-pantolactone dehydrogenase, D-keto-pantolactone reducase and glucose dehydrogenase are used as catalysts, the accumulation and spontaneous hydrolysis of the intermediate keto-pantolactone are avoided, the separation of intermediates, racemization of L-pantolactone and D-pantoic acid lactonization under acidic conditions are omitted, the reaction process is simplified, the use of acid-base 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, L-pantolactone dehydrogenase derived from Nocardiacyriacigeorgica, its coding 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 method catal...

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陈梁毛王伟殷杭华张连春白彦兵汪军姜伟林何敏王春霞郑世明
Owner HANGZHOU XINFU TECH CO LTD
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