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Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis

A technology for separation and purification of Bacillus subtilis, which is applied in the fields of fermentation and enzyme engineering, and can solve problems such as poor heat resistance, high impurity content in products, and high cost

Inactive Publication Date: 2013-07-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the problems of high cost, high product impurity content and poor heat resistance in the production of β-amylase from the above-mentioned plant sources, the present invention provides a fermented liquid of wild-type Bacillus subtilis 6-7 to efficiently separate and purify high-temperature-resistant β-amylase Methods

Method used

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  • Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis
  • Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis
  • Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis

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Experimental program
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Embodiment 1

[0034] Microorganism Bacillus subtilis B.subtilis6-7 is fermented to prepare fermentation broth crude enzyme liquid. The strain B.subtilis6-7 preserved in glycerol cryovials was activated and cultured in 10mL / 50mL LB medium for 12h, and then transferred to fresh 50mL / 250mL shake flask LB medium for 12h as secondary seeds. Optimized medium: 2% tapioca flour, 4% soybean meal flour, 0.1% diammonium hydrogen phosphate, 0.00139% ferrous sulfate, 0.6% sodium citrate, 0.4% potassium dihydrogen phosphate, 0.0005% zinc sulfate, 0.0123% magnesium sulfate, 0.0111% calcium chloride, pH 7.0. The volume of liquid in a 500mL shake flask is 250mL medium, sterilized at 121°C for 20min. The secondary seeds were inoculated in the fermentation optimization medium according to the inoculation amount of 4%. The rotation speed is 160r / min, and the culture temperature is 37°C for shaking flask culture. After 60 hours of fermentation and cultivation, the fermentation broth was collected, centrifuged...

Embodiment 2

[0036] Isolation and purification of microbial Bacillus subtilis B.subtilis6-7 thermostable β-amylase. First draw the ammonium sulfate salting-out curve. Take 7 bottles, respectively fill the same amount of 100ml crude enzyme solution, add different grams of ammonium sulfate solids according to the ammonium sulfate saturation table, respectively reach 10%, 20%, 30%, 40%, 50%, 60%, 70% % saturation, let it stand overnight, centrifuge to collect the precipitate, redissolve with pH 6.0 citrate buffer, measure the enzyme activity, and draw the salting-out curve with the initial relative enzyme activity as 100%. Add ammonium sulfate to the crude enzyme solution, according to the salting-out curve, centrifuge at 30% saturation to remove the impurity protein, collect the precipitate by centrifuging at 60% saturation, and precipitate the target protein β-amylase. Use pH8.0, 20mmol / L Na 2 HPO 4 -NaH 2 PO 4 Redissolve the precipitate in the buffer, put it into a dialysis bag with a...

Embodiment 3

[0038] Analysis of enzymatic properties of microbial Bacillus subtilis B.subtilis6-7 thermostable β-amylase. Optimum reaction temperature and temperature stability: measure the activity of β-amylase at 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80°C to determine the optimum reaction temperature; The enzyme solution was treated at different temperatures of 55, 60, 65, 70, and 75°C for different times, and the residual enzyme activity was determined. Optimum reaction pH and pH stability: Measure the activity of β-amylase in pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 citric acid-disodium hydrogen phosphate buffer solution to determine the optimum reaction pH; The enzyme solution was treated for different time at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and the residual enzyme activity was determined. Effect of metal ions and EDTA on enzyme activity: Add 1mmol / L Na to the enzyme reaction system + 、K + , Ca 2+ , Mg 2+ , Fe 2+ , Mn 2+ 、Ni 2+ 、Cu 2+ 8 kinds of metal ions and EDTA. And set a ...

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Abstract

The invention belongs to the technical field of bioengineering enzyme preparations and relates to a method for separation purification of high temperature-resistant beta-amylase from a wild-type bacillus subtilis 6-7 fermentation broth. Bacillus subtilis is preserved in the China center for type culture collection and has the preservation number of CTCC M2009200. The method is characterized in that wild-type B.subtilis6-7, and cassava powder and soybean meal as carbon and nitrogen sources are prepared into a fermentation broth; and the fermentation broth is subjected to refrigeration centrifugation, ammonium sulfate precipitation, dialysis desalting, HiTrap Qff ion column exchange, ultrafiltration centrifuge tube-based ultrafiltration desalting, monoQ5 / 50GL ion column exchange and Sephadex G-75 separation. The high temperature-resistant beta-amylase obtained by the method has a yield of 18.66%, purification fold of 4.83 and enzyme activity of 245395U / mg. An enzymatic property research shows that the purified high temperature-resistant beta-amylase has good heat resistance, is stable at the pH of 5-8 and is identified as the beta-amylase by mass spectrum identification. The high temperature-resistant beta-amylase has high purity, is colorless and satisfies food-grade beta-amylase standard requirements. The method provided by the invention has simple processes, is convenient for operation, has a high yield and realizes enzyme activity of 240000 U / mg.

Description

technical field [0001] The invention relates to a production process of wild-type Bacillus subtilis6-7 high temperature-resistant β-amylase, in particular to a method for efficiently separating and purifying a high-enzyme activity β-amylase from a fermentation broth, belonging to the field of fermentation and enzyme engineering . Background technique [0002] β-amylase (1,4-α-D-glucan maltohydrolase, EC3.2.1.2), is an exo-type glucoamylase, acting on starch or glycogen, from the non-reducing properties of α-1,4 glycosidic bonds The maltose unit is successively cut off at the end, and the hydrolysis products include maltose, maltotriose, β-limit dextrin and a small amount of glucose. Maltose undergoes Walden inversion at the same time, changing from α-type to β-type, so it is called β-amylase. In theory β-amylase can convert all of the amylose to maltose and about 60% of the amylopectin to maltose and the rest to dextrins. [0003] β-amylase is widely found in plants such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26
Inventor 饶志明邹艳玲徐美娟
Owner JIANGNAN UNIV