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Method of producing alkaline pectinase

A pectinase and alkaline technology, applied in the field of alkaline pectinase production, can solve the problem that the industrialized production technology of alkaline pectinase is immature, the production cost of alkaline pectinase is high, and the activity of alkaline pectinase can be solved. It can achieve the effect of low production cost, short fermentation cycle and high-efficiency extracellular expression.

Active Publication Date: 2013-09-25
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the following problems mainly exist in the industrialized production of alkaline pectinase: the activity of the produced alkaline pectinase itself is not high; ; The industrialized production technology of alkaline pectinase is immature; the above problems cause the production cost of alkaline pectinase to be too high

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  • Method of producing alkaline pectinase
  • Method of producing alkaline pectinase
  • Method of producing alkaline pectinase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the method for producing alkaline pectinase——IPTG-induced enzyme production method

[0045] 1. Preparation of culture medium

[0046] 1. Seed medium

[0047] The solvent is water, the solute and its final concentration in the seed culture medium are respectively: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, and the pH is 7.0.

[0048] 2. Fermentation medium

[0049] The solvent is water, the solute and its final concentration in the fermentation medium are respectively: glycerol 10.0g / L, KH 2 PO 4 10.5g / L, (NH 4 ) 2 HPO 4 6g / L, citric acid 1.7g / L, MgSO 4 1.78g / L,FeSO 4 70mg / L, ZnSO 4 29.5mg / L, CuSO 4 30mg / L, MnSO 4 5mg / L,Na 2 B 4 o 7 2.3mg / L, CaCl 2 20mg / L and (NH 4 ) 6 Mo 7 o 24 1mg / L; pH value is 7.0.

[0050] 3. Glycerol-fed medium

[0051] Solvent is water, and solute and its final concentration in described feed glycerin medium are respectively: glycerol 500g / L, MgSO 4 15.5g / L, peptone 15g / L, yeast powder 30g / L.

[0052] 2. Pr...

Embodiment 2

[0066] Embodiment 2, the method for producing alkaline pectinase——IPTG-induced enzyme production method

[0067] 1. Preparation of culture medium

[0068] 1. Seed medium

[0069] The solvent is water, the solute and its final concentration in the seed culture medium are respectively: peptone 5g / L, yeast extract 2g / L, NaCl 5g / L, and the pH is 6.9.

[0070] 2. Fermentation medium

[0071] The solvent is water, the solute and its final concentration in the fermentation medium are respectively: glycerol 8.0g / L, KH 2 PO 4 8g / L, (NH 4 ) 2 HPO 4 7g / L, citric acid 3g / L, MgSO 4 2.59g / L,FeSO 4 100mg / L, ZnSO 4 60mg / L, CuSO 4 40mg / L, MnSO 4 6mg / L,Na 2 B 4 o 7 3mg / L, CaCl 2 30mg / L and (NH 4 ) 6 Mo 7 o 24 2mg / L; pH value is 6.9.

[0072] 3. Glycerol-fed medium

[0073] Solvent is water, and solute and its final concentration in described feed glycerin medium are respectively: glycerol 400g / L, MgSO 4 7.76g / L, peptone 10g / L, yeast powder 20g / L.

[0074] 2. Preparation o...

Embodiment 3

[0088] Embodiment 3, the method for producing alkaline pectinase——IPTG-induced enzyme production method

[0089] 1. Preparation of culture medium

[0090] 1. Seed medium

[0091] The solvent is water, the solute and its final concentration in the seed culture medium are respectively: peptone 7g / L, yeast extract 3g / L, NaCl 8g / L, and pH 7.1.

[0092] 2. Fermentation medium

[0093] The solvent is water, the solute and its final concentration in the fermentation medium are respectively: glycerol 12.0g / L, KH 2 PO 4 12g / L, (NH 4 ) 2 HPO 4 5g / L, citric acid 0.5g / L, MgSO 4 1.55g / L,FeSO 4 50mg / L, ZnSO 4 45mg / L, CuSO 4 20mg / L,MnSO 4 4mg / L,Na 2 B 4 o 7 1mg / L, CaCl 2 15mg / L and (NH 4 ) 6 Mo 7 o 24 1mg / L.

[0094] 3. Glycerol-fed medium

[0095] Solvent is water, and solute and its final concentration in described feeding glycerol medium are respectively: glycerol 600g / L, MgSO 4 35g / L, peptone 20g / L, yeast powder 40g / L.

[0096] 2. Preparation of seed solution

[0...

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Abstract

The invention discloses a method of producing alkaline pectinase. According to the method, with Escherichia coli BL21 (DE3) PGL04CGMCC (China General Microbiological Culture Collection Center) NO.5697 as a starting strain, the efficient extracellular expression of the alkaline pectinase is realized through the following fermentation strategy and method: adopting an optimized batch fermentation culture medium to realize high density culture of the escherichia coli through a double-stage glycerol feeding strategy, and beginning to induce when the cell density reaches to a level of containing 10-15g / L of stem cells in each liter of fermentation broth, wherein the induction temperature is 28-30 DEG C; and adopting a constant speed feeding strategy of 2.5-7.5mL / (L.h) of 180-220g / L lactose solution to finally realize the efficient extracellular expression of the alkaline pectinase, and the extracellular enzyme activity can be higher than 4000U / mL. The method disclosed by the invention has the advantages of being simple in process, short in fermentation period and suitable for industrial production.

Description

technical field [0001] The invention relates to a method for producing alkaline pectinase. Background technique [0002] Alkaline pectinase (E.C.4.2.2.2) is a class of enzymes that cleave the main chain of pectin under alkaline conditions to produce oligomerized pectin fragments. Alkaline pectinase is mainly used in industrial fields such as biological scouring of cotton fabrics and hemp degumming to remove the pectin components of cotton and hemp. Compared with the traditional high-temperature and high-alkali chemical treatment method, the enzyme treatment technology based on alkaline pectinase has the advantages of mild conditions, environmental friendliness, low energy consumption, and easy clean production, and can also improve the quality of cotton fabrics . In addition, the enzyme can also be used in food processing, sewage treatment, paper and pulp and other industries. Therefore, efficient production of alkaline pectinase is of great significance to promote the in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12R1/19
Inventor 宋江宁王辉林李小曼马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI