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Marine microorganism fermentation production method for low temperature gamma-lactamase

A technology of marine microorganisms and lactamase, applied in the direction of hydrolytic enzymes, etc., to achieve the effect of simple operation, saving heating or cooling costs, and improving utilization rate and conversion rate

Inactive Publication Date: 2015-05-20
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no research reports on the production of low-temperature γ-lactamases by fermentation of marine microorganisms at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Culture medium preparation

[0019] ① Strain activation medium: yeast extract 20.0g, peptone 10.0g, NaCl 10.0g, agar 20.0g, tap water 1.0L, pH7.4, sterilized at 103kpa, 121℃ for 30min by high-pressure steam;

[0020] ② Low-temperature acclimation medium for strains: yeast extract 0.2g, N-acetyl-L-phenylalanine 1.0g, NH 4 Cl 2.0g, Na 2 HPO40.1g, NaH 2 PO40.1g, MgSO 4 0.05g, 20g agar powder, 1.0L tap water, pH 6.0, sterilize at 103kpa, 121℃ for 30min by high-pressure steam;

[0021] ③Liquid seed medium: yeast extract 20.0g, peptone 10.0g, NaCl 10.0g, MgSO 4 0.05g; ZnSO 4 ·7H 2 O2.4mg, tap water 1.0L, pH6.0, sterilized by high pressure steam at 103kpa, 121℃ for 30min;

[0022] ④ Liquid enzyme production medium: beef extract 10.0g, peptone 2.0g, acetamide 5.0g, Tween800.1g, (NH 4 ) 2 SO 4 1.0g, KH 2 PO 4 1.4g, MgSO 4 0.1g, CaCl 2 0.2g, ZnSO 4 ·7H 2 O 2.4mg, tap water 1.0L, pH value 5.0, 121°C, sterilize at 103kpa, 121°C high pressure steam for 30min.

...

Embodiment 2

[0030] (1) Culture medium preparation

[0031] ① Strain activation medium: yeast extract 20.0g, peptone 10.0g, NaCl 10.0g, agar 20.0g, tap water 1.0L, pH value 7.4, sterilized at 103kpa, 121℃ for 30min by high-pressure steam;

[0032] ②Low temperature acclimation medium for strains: 2.0g of yeast extract; 6.0g of N-acetyl-L-phenylalanine; NH 4 Cl 6.0g; Na 2 HPO 4 0.5g; NaH 2 PO 4 0.5g; MgSO 4 0.05g; agar powder 20g, tap water 1.0L, pH value 7.0, sterilized at 103kpa, 121℃ for 30min by high-pressure steam;

[0033] ③ Liquid seed medium: 20.0g yeast extract, 15.0g peptone, 12.0g NaCl, MgSO 4 0.01g; ZnSO 4 ·7H 2 O6.0mg, 1.0L tap water, pH value 7.0, sterilized by high pressure steam at 103kpa, 121℃ for 30min;

[0034] ④ Liquid enzyme production medium: beef extract 15.0g, peptone 15.0g, acetamide 15.0g, Tween800.3g, (NH 4 ) 2 SO 4 1.2g, KH 2 PO 4 1.6g, MgSO 4 0.3g, CaCl 2 0.2g, ZnSO 4 ·7H 2 O 6.0mg, tap water 1.0L, pH value 6.0, sterilized by high pressure steam...

Embodiment 3

[0042] (1) Culture medium preparation

[0043] ① Strain activation medium: yeast extract 20.0g, peptone 10.0g, NaCl 10.0g, agar 20.0g, tap water 1.0L, pH value 7.4, sterilized at 103kpa, 121℃ for 30min by high-pressure steam;

[0044] ②Low temperature acclimation medium for strains: yeast extract 5.0g; N-acetyl-L-phenylalanine 10.0g; NH 4 Cl 15.0g; Na 2 HPO 4 1.0g; NaH 2 PO 4 1.0g; MgSO 4 0.5g; agar powder 20g, tap water 1.0L, pH value 8.0, sterilized at 103kpa, 121℃ for 30min by high-pressure steam;

[0045] ③Liquid seed medium: yeast extract 25.0g, peptone 20g, NaCl 15.0g, MgSO 4 0.5g; ZnSO 4 ·7H 2 O8.0mg, 1.0L tap water, pH value 8.0, sterilized by high pressure steam at 103kpa, 121℃ for 30min;

[0046] ④ Liquid enzyme production medium: beef extract 25.0g, peptone 20.0g, acetamide 20g, Tween800.4g, (NH 4 ) 2 SO 4 1.5g, KH 2 PO 4 2.0g, MgSO 4 0.4g, CaCl 2 0.3g, ZnSO 4 ·7H 2 O 8.0mg, tap water 1.0L, pH value 8.0, sterilized by high pressure steam at 103kpa,...

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Abstract

The invention relates to a marine microorganism fermentation production method for low temperature gamma-lactamase. The method comprises the following steps: firstly activating the strain used for producing the gamma-lactamase, performing cold acclimation step by step and growing the strain in low temperature environment, performing the enlarged cultivation step by step on the cold acclimated gamma-lactamase producing strain at 10-16 DEG C, inoculating the producing strain to the liquid fermentation medium according to the ratio of 3-9% of the volume of the fermentation liquor, cultivating for 72-144h at 10-16 DEG C to obtain the low temperature gamma-lactamase, further concentrating, separating and purifying the collected crude enzyme liquid according to the different requirements and different use objects for preparing the enzyme preparation different in activity, purity and dosage form. The low temperature gamma-lactamase prepared by the method has high enzyme activity and high catalytic efficiency under a low temperature, the enzyme preparation is simple, convenient and fast in application operation, and broad industrial development value and market application advantage can be achieved.

Description

technical field [0001] The invention relates to the fields of microbiology, enzyme engineering, fermentation engineering, biochemistry and the like, and in particular relates to a method for producing low-temperature gamma-lactamase by fermentation of marine microorganisms. Background technique [0002] γ-lactamase (γ-lactamses, EC 3.5.2.-) is a novel amidase that catalyzes the hydrolysis of the amide bond of amide compounds. (+)γ-lactamase can kinetically resolve racemic γ-lactam with high efficiency to obtain optically pure (-)γ-lactam. Optically pure (-) γ-lactam is a chiral intermediate for the preparation of antiviral drugs. synthetic raw materials. In addition, γ-lactamase can catalyze the racemic γ-lactam to obtain γ-aminobutyric acid analogs (GABA) with different configurations. GABA is an inhibitory neurotransmitter, which participates in various metabolic activities and has high physiological activity (Wang Jianjun et al., Acta Microbiology, 2010). Before the d...

Claims

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Application Information

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IPC IPC(8): C12N9/86
CPCC12N9/86
Inventor 李晓艳王晓辉窦少华于爽张庆芳迟乃玉张旭姣王强金连豆
Owner DALIAN UNIV
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