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L-pantoyl lactone dehydrogenase derived from Nocardia farcinica and application of L-pantoyl lactone dehydrogenase

A pantolactone, ester dehydrogenase technology, applied in the application, enzyme, oxidoreductase and other directions, can solve the problems of unknown coding gene, hinder the application and other problems, reduce the use of acid and alkali, simplify the reaction process , to avoid the effect of separation

Active Publication Date: 2019-12-03
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] There is no report of L-pantolactone dehydrogenase derived from Nocardia farcinica, and no L-pantolactone dehydrogenase derived from Nocardia farcinica is used in multi-enzyme cascades to catalyze 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 Nocardia farcinica and application of L-pantoyl lactone dehydrogenase
  • L-pantoyl lactone dehydrogenase derived from Nocardia farcinica and application of L-pantoyl lactone dehydrogenase
  • L-pantoyl lactone dehydrogenase derived from Nocardia farcinica and application of L-pantoyl lactone dehydrogenase

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 Nocardia farcinica (GenBank accession number is BAD58055.1), 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 (NfaLPLDH) derived from Nocardia farcinica was inserted into the Hind III and Xho I sites of the plasmid pET-28b to obtain the recombinant plasmid pET-28b-NfaLPLDH. Transfer pET-28b-NfaLPLDH into E.coli BL21(DE3) to obtain genetically engineered bacteria E.coli BL21(DE3) / pET-28b-NfaLPLDH.

[0075] The genetically engineered bacteria E.coli BL21(DE3) / pET-28b-NfaLPLDH 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 the genetically engineered bacterium E.coli BL21(DE3) / pET-28b-NfaLPLDH 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 farcinica. The reaction system for dehydrogenating 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 (pH7.0). The reaction solution was added into a three-necked flask, and the reaction conditions were maintained at 30°C, 600rpm and pH7.0 under magnetic stirring, and the catalytic process was added dropwise with 1M Na 2 CO 3 The solution maintains a cons...

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 gene engineering, in particular to L-pantoyl lactone dehydrogenase and application of the L-pantoyl lactone dehydrogenase. The L-pantoyl lactone dehydrogenase derived from Nocardia farcinica is disclosed for the first time, has good enzymatic activity to L-pantolactone and can be applied to catalytic synthesis of D-pantolactone. A system for multi-enzyme cascade catalysis of chiral overturning of the L-pantolactone to synthesize the D-pantolactone is constructed, genetically engineered bacterium cells for co-expression of the L-pantoyl lactone dehydrogenase, D-ketopantolactone reductase and glucose dehydrogenase are taken as a catalyst, accumulation and spontaneous hydrolysis of an intermediate product, namely ketopantolactone, are avoided, the steps such as separation of the intermediate product, racemization of the L-pantolactone and lactonization of D-pantoic acid under the acid condition are omitted, the reaction process is simplified, using 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, and more specifically relates to L-pantolactone dehydrogenase derived from Nocardiafarcinica, 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 me...

Claims

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Application Information

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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张连春毛王伟陈梁白彦兵金文究沈汉军汪军蔡剑敏
Owner HANGZHOU XINFU TECH CO LTD
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