A sugar-resistant and acid-resistant β-glucosidase and its production method

A glucosidase and sugar-tolerant technology, applied in the biological field, can solve the problems of limited large-scale application and high price, and achieve the effects of large-scale production and use, good thermal stability and good tolerance

Active Publication Date: 2020-09-25
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

β-glucosidase is widely used, but due to the lack of industrialization in China, the high price limits its large-scale application. Therefore, once the low-cost and high-tolerance β-glucosidase is on the market, it will have huge market potential

Method used

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  • A sugar-resistant and acid-resistant β-glucosidase and its production method
  • A sugar-resistant and acid-resistant β-glucosidase and its production method
  • A sugar-resistant and acid-resistant β-glucosidase and its production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1Co 60 Mutagenesis and strain screening

[0022] Preparation of spore suspension: Take sterile physiological saline to wash the spores on the fresh slant of the original strain, transfer them into a triangular flask filled with glass beads, vibrate and disperse them on a shaker for 60 minutes to make a bacterial suspension, and carry out gradient Dilute, count on a hemocytometer and adjust the spore concentration to 10 6 A / mL or so.

[0023] co 60 Radiation mutagenesis: place a test tube containing an appropriate amount of spore suspension on a radiation plane with a certain dose rate, and use Co 60 Radiation mutagenesis was carried out with irradiation doses of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 kGy respectively. After treatment, gradient dilution was carried out, spread on a PDA medium plate, and cultivated at 30°C.

[0024] Determination of mutagen dose: make 10-fold serial dilutions of the mutagenized and non-mutated bacterial suspensions, draw 1mL o...

Embodiment 2

[0036] Example 2 Beta-glucosidase enzyme activity assay

[0037] Enzyme activity definition: the amount of enzyme required to release 1 μmol p-NP per minute is an enzyme activity unit U.

[0038] Determination method: centrifuge the fermentation broth at 3000r / min for 10min, and get the supernatant as the crude enzyme solution. Accurately absorb 0.1mL of the crude enzyme solution, add 0.9mL of disodium hydrogen phosphate-citric acid buffer solution with pH4.8, and place in a constant temperature water bath at 50°C Preheat for 5 minutes, add 1 mL of preheated 5 mmol / L p-NPG, react with a stopwatch at 50°C for 10 minutes, and immediately add 1 mL of 1 mol / L sodium carbonate solution to terminate the reaction. Absorbance was measured at 420 nm.

Embodiment 3

[0039] The glucose tolerance of embodiment 3β-glucosidase

[0040] Taking the supernatant of AN-4 fermented liquid obtained in Example 1 as a sample, add 0-18% glucose respectively in the reaction system, determine the activity of β-glucosidase according to the method described in Example 2, and obtain the relative enzyme activity change curve like figure 1 , when the glucose concentration is lower than 13%, it can enhance the enzyme activity, the highest enhancement is 10%, and at the glucose concentration of 14%, the enzyme activity still retains about 95%, indicating that the β-glucosidase produced by AN-1 has Higher glucose tolerance.

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a sugar-resistant and acid-resistant β-glucosidase and its production. The β-glucosidase is produced from Aspergillus niger AN-4, and the preservation number is CGMCC No.14139. The average shake flask fermentation activity of β-glucosidase resistant to high sugar concentration produced by Aspergillus niger AN-4 is above 523U / mL; It is relatively stable within the range; thermal stability is good, the optimum reaction temperature range is 45-65°C, and it still has 75% activity at 75°C for 1 hour; and it has good glucose tolerance. It has good effect in cellulose degradation, flavoring agent and other applications, and can be widely used in food, medicine, chemical industry and other industries.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a sugar-resistant and acid-resistant β-glucosidase and a production method thereof. Background technique: [0002] β-glucosidase, also known as β-D-glucoside glucohydrolase, is also known as gentiobiase, cellobiase and amygdalinase. β-glucosidase exists in many plants, insects, yeast, Aspergillus, Trichoderma and bacteria in nature. It belongs to the cellulase class and is an important component of the cellulolytic enzyme system. It is generally believed that at least three enzymes are required for the enzymatic action of cellulosic substances into glucose: endo-1,4-glucanase, exo-glucanase Cellobiohydrolase and β-glucosidase. Endoglucanases and exo-cellobiohydrolases degrade cellulose to cellobiose, which is then broken down to glucose by β-glucosidases. In this process, β-glucosidase plays a key role, and the content of β-glucosidase in the cellulase component is low, and its a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N1/14C12R1/685
CPCC12N9/2445C12Y302/01021C12N1/145C12R2001/685
Inventor 郭庆文张杰王兴吉盛花开钱娟娟
Owner SHANDONG LONGKETE ENZYME PREPARATION
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