Strain for highly producing acid-resistant high-temperature alpha-amylase and liquid fermentation method of strain

A technology of liquid fermentation and amylase, which is applied in the field of bioengineering, can solve the problems of poor thermal stability and narrow pH range, and achieve the effect of stable acid resistance and high temperature resistance

Active Publication Date: 2015-12-16
SHANDONG LONGKETE ENZYME PREPARATION
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0002] α-amylase is one of the most widely used enzyme preparations in industrial production. Amylases commonly used in the domestic market are generally medium-temperature α-amylase and high-temperature-resistant α-amylase, and their applicable pH is between 6-7. There are defects of poor thermal stability and narrow pH range, and there are limitations to the starch deep processing technology under acidic conditions

Method used

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  • Strain for highly producing acid-resistant high-temperature alpha-amylase and liquid fermentation method of strain
  • Strain for highly producing acid-resistant high-temperature alpha-amylase and liquid fermentation method of strain
  • Strain for highly producing acid-resistant high-temperature alpha-amylase and liquid fermentation method of strain

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Experimental program
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Effect test

Embodiment 1

[0026] The breeding of embodiment 1 bacterial strain

[0027] 1. Preparation of Bacterial Suspension

[0028] Draw 10 mL of Bacillus licheniformis B307 seed liquid into a centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, and wash the bacteria with 10 mL of normal saline. Centrifuge twice under the same conditions, and resuspend the precipitated bacteria with 2 mL of sterile water to obtain a bacterial suspension. The concentration of the bacterial suspension is 10 7 individual / mL.

[0029] 2. Atmospheric room temperature plasma mutagenesis

[0030] Pipette 10 μL of bacterial suspension onto the slide, place the slide on the rotary table in the operating room of the normal-pressure room temperature plasma system with tweezers, and slowly adjust the knob of the lifting platform (refer to the scale of the lifting platform) to make the distance between the slide and the generator about 2mm. Mutagenesis irradiation time selection 30s, 35s, 40s. After t...

Embodiment 2

[0034] Embodiment 2 liquid fermentation produces acid-resistant high-temperature α-amylase

[0035] Slant culture: take an inoculation loop of the Bacillus licheniformis mutant strain M6-X3 and inoculate it on a solid slope of LB, and culture at a constant temperature of 34° C. for 36 hours.

[0036] Shake flask culture: take an inoculation loop slant seed lawn, insert it into LB liquid medium, and cultivate it for 10 hours under the conditions of initial pH 6.6, 36°C, and shaker speed 250rpm.

[0037] Seed tank culture: the seed solution cultivated in shake flasks was transferred into the seed tank according to 4% of the inoculum amount, and cultivated for 13 hours under the conditions of initial pH 6.3, fermentation temperature 34°C, and rotation speed 250 rpm.

[0038] The formula of the seed tank culture medium is: 3% maltodextrin, 0.2% yeast powder, 0.25% calcium chloride, 0.8% ammonium sulfate, 0.5% peptone, 0.5% disodium hydrogen phosphate, pH6.3.

[0039] Fermentation...

Embodiment 3

[0044] The basic properties of the acid-resistant high-temperature α-amylase produced by embodiment 3

[0045] Based on the acid-resistant high-temperature α-amylase with an enzyme activity of 41518u / mL, the enzyme activity assay experiments were carried out at different temperatures and different pHs. The optimum reaction temperature of the enzyme produced by this strain is 96-100°C, and the optimum pH The range is 4.0-5.0. Incubate at 95°C for 3 hours, and the remaining enzyme activity is 95%.

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Abstract

The invention relates to Bacillus licheniformis for highly producing acid-resistant high-temperature alpha-amylase and a liquid fermentation method of Bacillus licheniformis. Bacillus licheniformis particularly is Bacillus licheniformis M6-X3, and the preservation number of the strain is CGMCC NO.10785; the fermentation activity of an induced strain of Bacillus licheniformis CGMCC NO.10785 in a method for producing cid-resistant high-temperature alpha-amylase by virtue of liquid fermentation is 38000u/mL-42000u/mL; the residual enzyme activity of produced acid-resistant high-temperature alpha-amylase is 95% after the temperature is preserved at 95 DEG C for 3h, and the optimal reaction temperature is 96-100 DEG C, the optimal reaction pH is 4.0-4.5; and Bacillus licheniformis has good high temperature resistance and acid resistance and can be widely applied to various industries including starch processing, beer brewing, fermentation, spinning and the like.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-temperature high-temperature amylase-producing bacterial strain and a liquid fermentation method thereof. Background technique: [0002] α-amylase is one of the most widely used enzyme preparations in industrial production. Amylases commonly used in the domestic market are generally medium-temperature α-amylase and high-temperature-resistant α-amylase, and their applicable pH is between 6-7. It has the defects of poor thermal stability and narrow pH range, and has limitations on the deep processing technology of starch under acidic conditions. [0003] Deep processing of starch products generally includes two processes: liquefaction and saccharification. Firstly, liquefaction requires gelatinization at 105°C to form starch paste, then liquefaction by amylase, and then saccharification by glucoamylase at a temperature range of 50°C-60°C; There are also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/28C12R1/10
Inventor 王兴吉吕彦茹刘文龙
Owner SHANDONG LONGKETE ENZYME PREPARATION
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