Strain capable of high yielding acid-resisting high-temperature alpha-amylase and application

A technology of acid resistance and amylase, applied in the field of bioengineering, can solve the problems of poor thermal stability and narrow pH range, and achieve the effect of stabilizing acid resistance and high temperature resistance.

Active Publication Date: 2021-02-23
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the optimum pH of the commercial acid-resistant high-temperature α-amylase widely used in domestic industry is generally around 5.0-6.0, most of which have the defects of poor thermal stability and narrow pH range, and have limitations in the deep processing of starch under acidic conditions.

Method used

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  • Strain capable of high yielding acid-resisting high-temperature alpha-amylase and application
  • Strain capable of high yielding acid-resisting high-temperature alpha-amylase and application
  • Strain capable of high yielding acid-resisting high-temperature alpha-amylase and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Mutagenesis and directional screening of embodiment 1 bacterial strain

[0041] The enzyme activity level of liquid fermentation of Bacillus licheniformis CGMCC NO.10785 needs to be further improved. In order to obtain a strain with high acid-resistant and high-temperature α-amylase, nitrosoguanidine mutagenesis was carried out until a strain with obvious growth changes was bred. The strain is fast and can be cultured in high density, and the enzyme activity level of acid-resistant and high-temperature α-amylase is further improved.

[0042] 1. Preparation of Bacterial Suspension

[0043] Take 300mL of Bacillus licheniformis CGMCC NO.10785 logarithmic phase seed liquid into six 50mL centrifuge tubes, centrifuge at 8000rpm for 5min, discard the supernatant, wash the precipitated bacteria twice with 600mL normal saline, and resuspend with 100-200mL normal saline It is the bacterial suspension.

[0044] 2. Nitrosoguanidine mutagenesis and coating screening plate

[0045...

Embodiment 2

[0053] Embodiment 2 liquid fermentation produces acid-resistant high-temperature α-amylase

[0054] Streak the seeds to isolate a single colony: Streak the frozen glycerol tube seeds of the Bacillus licheniformis mutant strain YBG-90036 on the seed slant medium to isolate a single colony, and culture at 37° C. for 30 h.

[0055] Seed slant medium formula: 4% corn starch, 3% yeast powder, 2% soybean peptone, 2% malt syrup, 0.4% calcium chloride, 1.5% ammonium chloride, 1.8% potassium dihydrogen phosphate, 1% agar powder, pH5.5.

[0056] Liquid seed culture: On the above-mentioned seed slant medium, pick a single colony with a high colony HC value (HC value = hydrolysis circle diameter / colony diameter), insert it into 200mL liquid seed medium (use a 500mL Erlenmeyer flask), 37°C , shaker speed 300 rpm / min, cultivate to OD 600nm The absorbance value is around 2.0.

[0057] Liquid seed medium: corn starch 4%, yeast powder 3%, soybean peptone 2%, malt syrup 2%, calcium chloride ...

Embodiment 3

[0065] The basic properties of the acid-resistant high-temperature α-amylase produced by embodiment 3

[0066] Under the conditions of 70°C and pH 4.0, the enzyme activity was measured, and the acid-resistant high-temperature α-amylase sample with an enzyme activity of 92040U / mL was used as the benchmark, and the samples were taken for enzyme activity determination experiments at different temperatures and different pHs. . The optimum pH judgment standard is that the enzyme activity measured in different pH buffer solutions is more than 80% of the enzyme activity of the sample under the condition of 70°C, which is the optimum pH range of the enzyme. The criterion for the optimum temperature range is that under the condition of pH 4.0, the enzyme activity measured at different temperatures is more than 120% of the enzyme activity of the sample, which is the optimum temperature range of the enzyme. The acid-resistant high-temperature α-amylase produced by the mutant strain YBG-...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to a strain capable of high yielding acid-resisting high-temperature alpha-amylase and application. The bacilluslicheniformis specifically is bacillus licheniformis YBG-90036 and has an accession number of CGMCC NO. 20672. Fermentation liquor for yielding the acid-resisting high-temperature alpha-amylase through liquid fermentation of the bacillus licheniformis mutagenic strain CGMCC NO. 20672 has the enzyme activity of 8.9U/mL to 9.2U/mL; after the yielded acid-resisting high-temperature alpha-amylase issubjected to heat preserving for 3h under the conditions that the pH is 4.0 and the temperature is 98 DEG C, the remaining enzyme activity is 98%; the optimal pH is 3.5-4.4; the optimal reaction temperature is 98 DEG C to 100 DEG C; and the acid-resisting high-temperature alpha-amylase has good high-temperature resistance and acid resistance and can be extensively applied to multiple industries such as starch processing, beer brewing, fermentation and spinning.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yield acid-resistant high-temperature amylase strain and a liquid fermentation method thereof. Background technique: [0002] α-amylase is one of the earliest commercially produced, most widely used and most used enzyme preparations. Indispensable in starch sugar and fermentation industry. [0003] Deep processing of starch products generally includes two processes: liquefaction and saccharification. First, adjust the pH value of the starch slurry from the natural 4.5 to 5.8-6.2, add high-temperature-resistant amylase and mix well, then heat it to 90°C with 105°C steam, and enzymatically hydrolyze it for 1-1.5h to form a starch paste. Then lower the pH value of the formed starch paste to about 4.0-4.5, lower the temperature to 50°C-60°C, and add glucoamylase for saccharification; the above two enzymes added not only have different action temperatures,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/28C12N15/01C12R1/10
CPCC12N15/01C12N9/2417C12Y302/01001C12R2001/10C12N1/205
Inventor 刘文龙佟新伟王兴吉钱娟娟刘胜利王克芬
Owner SHANDONG LONGKETE ENZYME PREPARATION
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