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Highly expressed Staphyloccocus aureus alpha-acetolacetate decearboxylase by utilization of recombinant escherichia coli

A technology of acetolactate decarboxylase and Escherichia coli is applied in the field of genetic engineering and enzyme engineering to improve production efficiency and shorten the aging cycle of beer

Inactive Publication Date: 2012-12-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme has not been found in eukaryotes such as fungi, algae and protozoa

Method used

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  • Highly expressed Staphyloccocus aureus alpha-acetolacetate decearboxylase by utilization of recombinant escherichia coli
  • Highly expressed Staphyloccocus aureus alpha-acetolacetate decearboxylase by utilization of recombinant escherichia coli
  • Highly expressed Staphyloccocus aureus alpha-acetolacetate decearboxylase by utilization of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Construction and transformation of recombinant plasmid pET-28a-saald

[0022] [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.

[0023] [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...

Embodiment 2

[0026] Example 2: Expression of α-acetolactate decarboxylase and assay of enzyme activity

[0027] [1] Inoculate the strains stored in cryopreserved tubes in Example 1[4] into 10 mL of liquid LB medium, cultivate overnight at 37°C with shaking, transfer at 1% inoculum size the next day, and cultivate to OD at 37°C 600Add IPTG at a final concentration of 1 mmol / L at about 0.6 to 0.8, and place on a shaker at 30°C overnight to induce expression. Take 1mL of the obtained bacterial solution in a 1.5mL centrifuge tube, centrifuge at 8000r / min for 2min, pour off the supernatant, add 100L Tris-HCl buffer solution with pH 6.8 to mix the bacteria, add 30L protein gel loading buffer, boil water Bath for 30 minutes to denature the protein, centrifuge at 8000r / min for 2 minutes, take the supernatant for SDS-PAGE detection, wherein SDS-PAGE uses 5% separating gel and 15% stacking gel, adopts discontinuous vertical electrophoresis, and uses Coomassie brilliant blue R250 For staining, E.coi...

Embodiment 3

[0029] Example 3: Purification and Enzymatic Properties of α-Acetolactate Decarboxylase

[0030] [1] Pure ALDC was obtained after the crude enzyme solution was purified by Ni-NTA column. The specific activity of the purified enzyme solution was 469.85U / mg, the purification factor was 8.36 times, and the recovery rate was 87.65%.

[0031] [2] Optimum pH and pH stability: Prepare substrate solutions with different pH (2-12) with substrate buffer MES II, dilute the enzyme solution with MES I to a certain number of times, and then mix 380 μL of substrates with different pH with 20 μL of the diluted enzyme solution was reacted, and the enzyme activity under different pH conditions was measured and compared to determine the optimum pH value of the enzyme reaction. Prepare enzyme activity assay buffer MES I with different pH (5-10), and use them to dilute the enzyme solution to obtain enzyme solution with different pH. Let the enzyme be in different pH environments, take samples at ...

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Abstract

A staphylococcus aureus genome DNA is used as a template to be amplified so as to obtain a gene saald for encoding alpha-acetolacetate decearboxylase (ALDC). The gene is cloned to an escherichia coli expression vector pET-28a and inducible expression is carried out on E.coli BL21 by IPTG. By the utilization of 6.His-Tag on the expression vector pET-28a, a Ni column is selected for purification and expression of the active alpha-acetolacetate decearboxylase by affinity chromatography. Specific enzyme activity of crude enzyme is 56 U / mg; specific enzyme activity reaches 469.85 U / mg after purification and the purification multiple reaches 8.36 times; and recovery rate is 87.65%.

Description

technical field [0001] The invention relates to highly expressing Staphyloccocus aureus α-acetolactate decarboxylase by using recombinant Escherichia coli and belongs to the fields 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. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N9/88C12R1/19C12R1/445
Inventor 饶志明李静静张显徐美娟
Owner JIANGNAN UNIV
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