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Leucine dehydrogenase as well as preparation method and application thereof

A leucine dehydrogenase and amino acid technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effects of reducing production costs, prolonging half-life, and improving production efficiency

Active Publication Date: 2015-07-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the Leucine dehydrogenase gene of the high-temperature Actinosaccharomyces leucine. In view of the advantages of the leucine dehydrogenase in catalyzing the preparation of L-leucine, tert-leucine, etc., the selection Highly active leucine dehydrogenase strain is of great significance for industrial production

Method used

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  • Leucine dehydrogenase as well as preparation method and application thereof
  • Leucine dehydrogenase as well as preparation method and application thereof
  • Leucine dehydrogenase as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment l

[0041] Embodiment 1: Containing the construction of the recombinant plasmid pET22b-LeuDH of leucine dehydrogenase gene sequence

[0042] Using the database of the National Center for Biotechnology Information (NCBI), we analyzed, screened, and found that there is a sequence in Laceyella sacchari that has a high similarity to the sequence of the leucine dehydrogenase gene, which is likely to encode the leucine dehydrogenase The gene sequence is screened and optimized (optimization includes mutation and insertion of some sites) to obtain the nucleotide sequence with SEQ ID NO.2.

[0043] The Ls-LeuDH gene was artificially synthesized by GenScript according to the nucleotide sequence of SEQ ID NO.2. Add Nde I (CAT) and Xho I (CTCGAG) at both ends of the sequence, respectively, and connect them between the Nde I and Xho I restriction sites on the pET22b plasmid vector. Get the recombinant plasmid pET22b-LeuDH, transfer it into 50 microliters of Escherichia coli competent c...

Embodiment 2

[0045] Example 2: Expression and purification of recombinant leucine dehydrogenase

[0046] Pick engineering bacteria with a single colony E. coli BL21-pET22b-LeuDH, cultured in 10ml LB liquid medium containing 100μg / mL ampicillin with shaking at 37°C for 10 hours; pipette the culture solution according to the inoculum size of 1v / v% and transferred to Cultured at 30°C in a self-inducing liquid medium (the formulation of the self-inducing medium is (per 100 ml): 1.5 grams of peptone, 2.5 grams of yeast powder, 1 gram of sodium chloride, 0.2 grams of glucose, and 0.3 grams of lactose.), to be OD 600nm After reaching 1.0, incubate at 25°C for 20 hours at low temperature, collect the bacterial solution by centrifugation, discard the supernatant medium, wash with equal volume of PBS buffer with pH=8 for three times, add PBS buffer to make the resuspended bacterial solution OD 600nm When it reaches 20, mix well; break with an ultrasonic breaker, ultrasonic working conditio...

Embodiment 3

[0047] Example 3: Determination of Leucine Dehydrogenase Enzyme Activity.

[0048] Each component was added according to the reaction system shown in Table 1, and the expression product of Escherichia coli containing plasmid pET22b was used as a control. The basic principle is that in view of the fact that NADH has a maximum absorption peak at 340nm, the content of the enzyme is quantitatively determined by the change of the light absorption peak. Add in sequence to the cuvette

[0049] Reaction solution 1.95ml: Na at pH 10.0 2 CO 3 / NaHCO 3 Buffer with 10mM L-Leucine, 3.5mM NAD + . Place it in the sample tank of a spectrophotometer, detect the original NADH absorbance value, and immediately add 50 μL of enzyme solution diluted with an appropriate multiple after the reading is stable, start the photometer reading, measure the absorbance value every 1 s, for a total of 30 s, and get △ A 340nm / min value. The enzyme activity unit adopts the international unit IU, that...

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Abstract

The invention discloses a leucine dehydrogenase as well as a preparation method and application thereof. The leucine dehydrogenase is encoded by taking Laceyella sacchari genome as a basis and cloning the leucine dehydrogenase gene LeuDH thereof, wherein the amino acid sequence is shown in SEQ ID NO. 1. The invention further discloses a gene for encoding the leucine dehydrogenase, and the nucleotide sequence is shown in SEQ ID NO. 2. The invention provides a novel dehydrogenase different from the present existing leucine dehydrogenase in sources. The leucine dehydrogenase is good in heat-resistant property, wide in pH application range, and capable of effectively prolonging a half-life period and reducing the production cost in industrial production.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and microorganisms, in particular to a leucine dehydrogenase and its preparation method and application. Background technique [0002] Leucine Dehydrogenase (Leucine Dehydrogenase, LeuDH, EC1.4.1.9) is an oxidoreductase with NADH and its oxidation state as coenzyme factors. The enzyme can catalyze the oxidation of L-leucine and other branched-chain L-type amino acids, and has the ability to reduce its corresponding ketoacids to L-type amino acids. The enzyme was first screened from Bacillus subtilis, and later gradually found to exist in Bacillus, Bacillus, Corynebacterium, and Thermoactinomycetes. The current highly active leucine dehydrogenase strains have been screened out and used in industrial production, such as: Lysinibacillus sphaericus, Bacillus cereus and Exiguobacterium sibiricum) etc. [0003] Due to the high thermal stability of thermophilic enzymes in industrial ap...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/04
CPCC12N9/0016C12P13/04C12Y104/01009
Inventor 贾红华韦萍朱文骏
Owner NANJING UNIV OF TECH
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