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Method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation

A fed-batch fermentation and pretreatment technology, which is applied in the field of production of alkaline pectinase with high enzyme activity, can solve the problems of low activity of alkaline pectinase, unsuitability for industrial production, and high cost, so as to shorten the fermentation cycle and improve flow rate. Sexuality and oxygen carrying capacity, the effect of improving quality

Active Publication Date: 2013-07-03
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the prior art, the alkaline pectinase produced by liquid fermentation has low activity, high cost and is not suitable for industrialized production. pectinase method

Method used

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  • Method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation
  • Method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation

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Experimental program
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Effect test

Embodiment 1

[0030] Bacillus subtilis CCTCC NO:M200038 (applicant deposited in China Center for Typical Cultures (CCTCC) on November 20, 2000, see ZL01106844.2 for related patents) was activated on an inclined plane, and the bacterial lawn was inoculated to the medium containing the seeds The shake flasks were cultured under the following conditions: 300ml Erlenmeyer flask with 50ml liquid volume, fermentation temperature 37°C, 200rpm shaking culture for 8h. Inoculate the initial fermentation medium with an inoculum size of 10%, at a temperature of 33-34°C, and an aeration volume of 2.5-4.5L min -1 . The starch in the feed medium was liquefied with mesophilic amylase, and the amount of enzyme added was 25U g -1 , the liquefaction temperature is 60°C, and the liquefaction time is 35min. After the initial fermentation to 14 hours, add the feed medium after pretreatment according to 20% of the volume of the initial fermentation medium, control the temperature at 33-34°C, and the ventilation...

Embodiment 2

[0035] Fermentation method, condition are all the same as embodiment 1 except following changes. In the pretreatment process, the amount of enzyme added by mesophilic amylase is 32.5U g -1 , the liquefaction temperature is 55°C, and the liquefaction time is 30min. Primary fermentation to 16h feeding. The fermentation period is 36-48h, and the highest alkaline pectinase activity of the obtained crude enzyme solution is 531U mL -1 . Fermentation see figure 1 . Wherein the culture medium formula is as follows:

[0036] The composition of the seed medium is: glucose 11g L -1 , yeast powder 4,5g L -1 , peptone 4.5g L -1 , NaCl 4.5g L -1 , K 2 HPO 4 10gL -1 , pH 8.0;

[0037] The composition of the fermentation medium is: starch 20g L -1 , wheat bran 40g L -1 , (NH 4 ) 2 SO 4 1.5gL -1 , MgSO 4 ·7H 2 O 1gL -1 ;

[0038] The feed medium composition is: starch 122.5g L -1 , wheat bran 10.5g L -1 , (NH 4 ) 2 SO 4 10gL -1 , MgSO 4 ·7H 2 O6.5gL -1 .

Embodiment 3

[0040] Fermentation method, condition are all the same as embodiment 1 except following changes. In the pretreatment process, the amount of amylase added in the middle temperature is 40U g -1 , the liquefaction temperature is 50°C, and the liquefaction time is 25min. Primary fermentation to 18h feeding. The fermentation period is 36-48h, and the final alkaline pectinase activity of the obtained crude enzyme solution is 479UmL -1 . Fermentation see figure 1 . Wherein the culture medium formula is as follows:

[0041] The composition of the seed medium is: glucose 13g L -1 , yeast powder 6g L -1 , peptone 6g L -1 , NaCl 6g L -1 , K 2 HPO 4 12gL -1 , pH 8.0;

[0042] Above-mentioned fermentation medium composition is: starch 25g L -1 , wheat bran 45g L -1 , (NH 4 ) 2 SO 4 2g L -1 , MgSO 4 ·7H 2 O 1.5g L -1 ;

[0043] The composition of the above-mentioned feeding medium is: starch 130g L -1 , wheat bran 13g L -1 , (NH 4 ) 2 SO 4 14g L -1 , MgSO 4 ·...

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Abstract

The invention discloses a method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation. The method is implemented by liquefying treatment and feed-batch fermentation for starch in a feed culture medium. The method obviously increases unit volume enzyme activity and volume production efficiency of the pectate lyase. Due to enzyme liquefaction treatment to the starch in the feed culture medium, viscosity coefficients of liquid are greatly reduced, and stirring efficiency is improved. Logarithmic growth phase of fungi is prolonged by means of feed fermentation, and biomass liveweight is increased. Experiments show that fermentation coarse enzyme fluid of the pectate lyase with the poly galacturonic acid cracking enzyme activity of 531UmL-1 is obtained by the method, and the highest volume production efficiency reaches 13U(mL*h)-1. The method further has the advantages of low cost, short fermenting period, high equipment utilization rate, simple process and suitability for large-scale industrial production.

Description

technical field [0001] The invention relates to a method for producing alkaline pectinase with high enzyme activity, in particular to a method for producing alkaline pectinase with high enzyme activity by performing liquefaction treatment and fed batch fermentation on starch in feed medium , belongs to the technical field of biological enzyme preparation. Background technique [0002] Pectinase (pectinases) is a general term for a class of enzymes that can decompose pectin, a component of plant cell walls. From the perspective of decomposition methods, pectinases can be divided into pectinases and pectin lyases. The former mainly includes protopectin enzyme (PPase), pectin esterase (PE), oligogalacturonase, polymethylgalacturonase (PMG), polygalacturonase (PG); the latter mainly includes polygalacturonase Lacturonate lyase (PGL) and polymethylgalacturonate lyase (PMGL). Alkaline pectinase, which is often called by people, generally refers to polygalacturonic acid lyase in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12R1/125
Inventor 邹谋勇李雪芝赵建曲音波
Owner SHANDONG UNIV
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