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Method for producing low-temperature protease by microbial fermentation

A low-temperature protease and microbial fermentation technology, applied in the field of low-temperature protease microbial fermentation production, can solve the problems that have not been reported (Wang Haiting et al., Marine Aquatic Research, 2002; Zhao Yuanyuan et al., Daily Chemicals Science, still in its infancy) and other issues

Inactive Publication Date: 2011-06-15
DALIAN UNIVERSITY
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Problems solved by technology

The research on low-temperature protease is still in its infancy, mainly focusing on strain selection, fermentation condition optimization, enzymatic properties and related gene cloning (Wang Quanfu et al., China Fishery Science, 2005; Chi Naiyu et al., Microbiology Bulletin, 2006; Zhang Xiu et al., China Fishery Science, 2006; Feng Xiuping et al., China Biotechnology Journal, 2009); and the industrialization, large-scale production and application of low-temperature protease have not yet been reported (Wang Haiting et al., Marine Aquatic Research, 2002; Zhao Yuanyuan et al., Science of Daily Chemicals, 2005)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] (1) Culture medium preparation

[0013] ① Strain activation medium: beef extract 0.3%, peptone 1.0%, NaCl 0.5%, casein 2%, agar 2%, tap water 1000ml, pH7.4, 0.1Mpa, sterilized at 121°C for 20min.

[0014] ②Low-temperature acclimation medium for strains: beef extract 0.1%-0.5%, peptone 0.7%-1.2%, NaCl 0.4%-0.6%, agar 2%, casein 1%-4%, tap water 1000ml, pH 7.4, 0.1Mpa , sterilized at 121°C for 20 minutes.

[0015] ③Liquid seed medium: glucose 0.8%~1.2%, peptone 0.3%~0.7%, yeast extract 0.3%~0.8%, K 2 HPO 4 0.08%~0.12%, MgSO 4 0.01%~0.04%, Na 2 CO 3 0.8%~1.2%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0016] ④Enzyme production medium: glucose 0.9%~1.3%, peptone 0.2%~0.6%, yeast extract 0.3%~0.7%, K 2 HPO 4 0.07%~0.13%, MgSO 4 0.01%~0.03%, Na 2 CO 3 0.8%~1.3%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0017] (2) The strains producing protease are initially activated according to the strain instructions provided by the China General Microorganism Cu...

Embodiment 2

[0024] (1) Culture medium preparation

[0025] ① Strain activation medium: beef extract 0.3%, peptone 1.0%, NaCl 0.5%, casein 2%, agar 2%, tap water 1000ml, pH7.4, 0.1Mpa, sterilized at 121°C for 20min.

[0026] ②Low-temperature acclimation medium for strains: beef extract 0.1%-0.5%, peptone 0.7%-1.2%, NaCl 0.4%-0.6%, agar 2%, casein 1%-4%, tap water 1000ml, pH 7.4, 0.1Mpa , sterilized at 121°C for 20 minutes.

[0027] ③Liquid seed medium: glucose 0.8%~1.2%, peptone 0.3%~0.7%, yeast extract 0.3%~0.8%, K 2 HPO 4 0.08%~0.12%, MgSO 4 0.01%~0.04%, Na 2 CO 3 0.8%~1.2%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0028] ④Enzyme production medium: glucose 0.9%~1.3%, peptone 0.2%~0.6%, yeast extract 0.3%~0.7%, K 2 HPO 4 0.07%~0.13%, MgSO 4 0.01%~0.03%, Na 2 CO 3 0.8%~1.3%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0029] (2) The strains producing protease are initially activated according to the strain instructions provided by the China General Microorganism Cu...

Embodiment 3

[0036] (1) Culture medium preparation

[0037] ① Strain activation medium: beef extract 0.3%, peptone 1.0%, NaCl 0.5%, casein 2%, agar 2%, tap water 1000ml, pH7.4, 0.1Mpa, sterilized at 121°C for 20min.

[0038]②Low-temperature acclimation medium for strains: beef extract 0.1%-0.5%, peptone 0.7%-1.2%, NaCl 0.4%-0.6%, agar 2%, casein 1%-4%, tap water 1000ml, pH 7.4, 0.1Mpa , sterilized at 121°C for 20 minutes.

[0039] ③Liquid seed medium: glucose 0.8%~1.2%, peptone 0.3%~0.7%, yeast extract 0.3%~0.8%, K 2 HPO 4 0.08%~0.12%, MgSO 4 0.01%~0.04%, Na 2 CO 3 0.8%~1.2%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0040] ④ Enzyme production medium: 0.9% to 1.3% glucose, 0.2% to 0.6% peptone, 0.3% to 0.7% yeast extract, K 2 HPO 4 0.07%~0.13%, MgSO 4 0.01%~0.03%, Na 2 CO 3 0.8%~1.3%, pH 8.5, 0.1Mpa, sterilized at 121°C for 20min.

[0041] (2) The strains producing protease are initially activated according to the strain instructions provided by the China General Microo...

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Abstract

The invention relates to a method for producing low-temperature protease by microbial fermentation, which comprises the following steps: carrying out low-temperature domestication on protease-producing microbes step by step, so that the microbes can well grow in a low temperature environment; carrying out amplification culture on the protease-producing microbes, which are subjected to low-temperature domestication, at 14-20 DEG C step by step; inoculating the protease-producing microbes, which account for 3-9% of a fermentation liquid by volume, into a protease-producing culture medium; culturing at 14-20 DEG C for 48-120 hours, and finishing the production of the low-temperature protease by microbial fermentation; centrifugating the fermentation liquid at 4000-8000 rpm, and collecting the liquid which is a crude enzyme liquid; and according to different demands and different use objects, further concentrating the crude enzyme liquid, and purifying by separation to obtain enzyme preparations with different activities, purities and forms.

Description

technical field [0001] The invention relates to the fields of microbiology, enzyme engineering, fermentation engineering, biochemistry and the like, in particular to a low-temperature protease microbial fermentation production method. The low-temperature protease produced by the invention is mainly used in feed industry, food processing, beer brewing, dairy product production, cosmetics industry, printing and dyeing, washing, leather manufacturing, environmental protection and other protease application industries. It can improve the utilization rate, conversion rate and productivity of raw materials, reduce production costs, and improve and enhance product quality. Background technique [0002] Protease is a general term for a class of enzymes that hydrolyze peptide bonds in proteins. As an important class of industrial enzymes, protease has been widely used in feed industry, food processing, beer brewing, dairy production, cosmetics industry, printing and dyeing, washing,...

Claims

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Application Information

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IPC IPC(8): C12N9/50
Inventor 张庆芳迟乃玉马莉
Owner DALIAN UNIVERSITY