Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently

A technology of acetolactate decarboxylase and Bacillus subtilis, which is applied in the field of genetic engineering and enzyme engineering, to shorten the aging cycle and improve production efficiency

Inactive Publication Date: 2013-01-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The enzyme has not been found in eukar

Method used

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  • Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently
  • Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently
  • Method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently

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Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: Construction and transformation of recombinant plasmid pMA5-saald

[0023] [1] Extract chromosomal DNA from Staphylococcus aureus as a template, the extraction method is as follows: use an inoculation loop to pick a single colony from a fresh Staphylococcus aureus plate and suspend it in 0.5mL sterilized double distilled water, boil it for 5 -10min, centrifuge at 8000r / min for 10min, take the supernatant into a clean 1.5mL centrifuge tube, and store in a -20°C refrigerator for later use.

[0024] [2] Using the total DNA of Staphylococcus aureus as a template, use the primers provided in Example 1 for PCR amplification. The amplification conditions are: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 56°C annealing, 1min, 72°C extension, 1min30s, 30 cycles; 72°C, 10min, one cycle; 15°C, 10min, one cycle. PCR amplification system: template (Staphylococcus aureus chromosomal DNA) 2 μL, upstream and downstream primers 0.5 μL each, dNTP Mix 4 ...

Embodiment 2

[0028] Example 2: Expression of α-acetolactate decarboxylase and assay of enzyme activity

[0029] [1] Inoculate the recombinant bacterium pMA5-saald / B.subtilis WB600 constructed in Example 1[5], the starting bacterium S.aureus and the host bacterium B.subtilis WB600 into 10 mL of liquid LB medium containing kanamycin, respectively medium, 37°C shaker at 160r / min shaking culture overnight, the next day transfer according to 1% inoculum size, culture at 37°C for 24h, take 1mL of the obtained bacterial solution in a 1.5mL centrifuge tube, centrifuge at 8000r / min for 2min, pour off the above Add 100 μL Tris-HCl buffer solution of pH 6.8 to mix the cells, add 30 μL protein gel loading buffer, bathe in boiling water for 30 minutes to denature the protein, centrifuge at 8000r / min for 10 minutes, and take the supernatant for SDS-PAGE detection , wherein SDS-PAGE uses 5% separating gel and 15% stacking gel, adopts discontinuous vertical electrophoresis, stains with Coomassie brilliant...

Embodiment 3

[0031] Embodiment 3: the fermentation of recombinant bacterium pMA5-saald / B.subtilis WB600

[0032] After the recombinant bacterium pMA5-saald / B.subtilisWB600 constructed in Example 1 [5] was activated and cultivated through LB medium, the fermentation medium was inoculated according to an inoculum size of 4% (soybean peptone 10g / L, corn steep liquor 15g / L, urea 3g / L, glucose 50g / L, k 2 HPO 4 ·3H 2 O 1.7g / L, kH 2 PO 4 2.3g / L, MgSO 4 ·7H 2 (0.75g / L, NaCl 5g / L, pH 6.8), 37 DEG C of shaker 160r / min culture 48h, finish fermentation, carry out the mensuration of enzyme activity according to the method described in embodiment 2 [2] then, its extracellular, The intracellular enzyme activities were 28300U / L and 38200U / L respectively, and the total enzyme activity reached 66500U / L.

[0033]

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Abstract

The invention relates to a method for expressing staphyloccocus aureus alpha-acetolactate decarboxylase by utilizing recombinant bacillus subtilis efficiently. The method comprises the following steps of: performing amplification by taking genomic deoxyribonucleic acid (DNA) of a staphyloccocus aureus geneome to obtain a gene saald for encoding alpha-acetolactate decarboxylase (ALDC); and cloning the gene saald on a shuttle expression vector pMA5, and constructing an alpha-acetolactate decarboxylase genetic engineering bacterium pMA5-saald/B.subtilis WB600 by taking B.subtilis WB600 as an expression host to realize the high-efficiency expression of ALDC. The fermenting enzyme activity of the engineering bacterium in a Luria-Bertani culture medium is 36,000 U/L, is improved by about 1,200 times compared with an original strain S. aureus and is improved by about 10,588 times compared with a host strain B. subtilis WB600. The recombinant bacterium is fermented and cultured in the fermentation culture medium for 48 hours, the enzyme activity of the ALDC is 66,500 U/L.

Description

technical field [0001] The invention relates to highly expressing Staphyloccocus aureus α-acetolactate decarboxylase by using recombinant subtilis bacillus, which belongs to the field of genetic engineering and enzyme engineering. Background technique [0002] α-acetolactate decarboxylase (ALDC for short) can catalyze the decarboxylation of diacetyl precursor α-acetolactate to form acetoin, avoiding the formation of diacetyl, which can shorten the beer aging cycle and improve production efficiency , of great economic value to the beer industry. [0003] α-Acetolactate decarboxylase is mainly used in beer brewing industry. In the metabolic process of brewer's yeast, α-acetolactate is an intermediate product in the biosynthesis of leucine-valine. Most of the α-acetolactate is metabolized in the yeast cell to form valine and leucine, and a small part leaks out of the cell, enters the fermentation broth, and generates diacetyl through non-enzymatic oxidation outside the cell. ...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N9/88C12R1/125C12R1/445
Inventor 饶志明李静静张显徐美娟杨套伟
Owner JIANGNAN UNIV
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