CN103981125B - The Geobacillus stearothermophilus bacterial strain of cyclodextrin glycosyltransferase is produced in one strain - Google Patents

Abstract

The present invention relates to a strain and produce the Geobacillus stearothermophilus bacterial strain of cyclodextrin glycosyltransferase, bacterial strain is stearothermophilus ground bacterial strain of bacillus (Geobacillus caldoxylosilyticus) CHB1, this bacterial strain on August 28th, 2013 in the registration preservation of Chinese Typical Representative culture DSMZ, deposit number is CCTCC M 2013384, and proving that the produced enzyme of this Geobacillus stearothermophilus is the cyclodextrin glucosyltransferase (being called for short CGTase) being subordinate to alpha amylase family, produced CGTase can be catalyzed soluble starch and generate α cyclodextrin.It is an advantage of the current invention that: provide the bacterial strain Geobacillus sp CHB1 of a kind of new product CGTase, and it is 65 DEG C that this bacterium produces enzyme fermentation liquid supernatant enzyme optimal reactive temperature alive, activity is kept between 40 DEG C ~ 85 DEG C, the produced CGTase of this bacterium can be used to produce α cyclodextrin, applies also for the course of processing of the starch etc. of this temperature range.

Description

The Geobacillus stearothermophilus bacterial strain of cyclodextrin glycosyltransferase is produced in one strain

Technical field

The present invention relates to a kind of Geobacillus stearothermophilus and application thereof, particular, it is found that a kind of new product high temperature cyclodextrin Glucosyltransferase (CGTase) bacterial strain Geobacillus sp and apply this strain fermentation produce cyclodextrin glucosylation Enzyme, belongs to fermenting and producing field.

Background technology

Cyclodextrin glycosyltransferase is the kind of starch enzyme that can change into cyclodextrin with catalytic starch, belongs to α-shallow lake The important member of powder enzyme family, it has cyclisation, is disproportionated, couples and hydrolyzes four kinds of activity.Main its cyclization of application in production Generating cyclodextrin, cyclodextrin glucosyltransferase catalysate is tri-kinds of cyclodextrins of α, β and γ, respectively by 6,7 and 8 glucose Glycan molecule joins end to end with α-1.4 glycosidic bond and forms.Define cyclodextrin glucosyltransferase with principal product difference can be divided into again Alpha-cyclodextrin glucosyltransferase, beta-schardinger dextrin-glucosyltransferase and gamma-cyclodextrin glucosyltransferase.The outside parent of cyclodextrin The cavity structure of water inner hydrophobic can play slow release in conjunction with multiple small-molecule substance, cover the multi-effect such as abnormal smells from the patient, protection, Important application is had in fields such as medicine, food, cosmetics and agriculturals.The production of cyclodextrin mainly application CGTase is catalyzed Arriving, the CGTase of current domestic application and report produces bacterial strain and mostly is the room temperature bacterium such as Bacillus genus, and produced CGTase is also normal Temperature enzyme.High temperature enzyme has the advantage that the room temperature enzymes such as catalytic rate is fast, tolerable temperature is high are incomparable.The inventors discovered that a strain is produced The bacterial strain of high temperature CGTase, and there is no other bacterial strain of the same race of Geobacillus caldoxylosilyticus at present and produce this enzyme Report, obtains crude enzyme liquid by liquid fermentation, and this crude enzyme liquid can produce cyclodextrin with efficient catalytic soluble starch.More than based on Discovery proposes the present invention.

Summary of the invention

It is an object of the invention to provide a strain and produce the Geobacillus stearothermophilus bacterial strain of cyclodextrin glycosyltransferase, This bacterial strain is Geobacillus stearothermophilus CHB1, is accredited as Geobacillus caldoxylosilyticus, named Geobacillus caldoxylosilyticu CHB1, this bacterial strain on August 28th, 2013 at Chinese Typical Representative culture strain The registration preservation of preservation center, deposit number is CCTCC M 2013384.And generate CGTase crude enzyme liquid with this strain fermentation, should Principal product alpha-cyclodextrin, optimal reaction temperature can be generated at high temperature (40 DEG C～85 DEG C) condition catalysis soluble starch with this enzyme liquid Degree is 60 DEG C～65 DEG C.

The present invention implements by the following technical programs:

First laboratory is separated to a strain at Foochow one and produces dextrin glucosyltransferase in the soil of hen house compost Bacterial strain CHB1, identified after concrete Classification And Nomenclature be Geobacillus caldoxylosilyticus CHB1, culture presevation in On August 28th, 2013, deposit number was CCTCC M 2013384 in the registration preservation of Chinese Typical Representative culture DSMZ, Preservation address is Wuhan University.

Tool with described bacterial strain Geobacillus caldoxylosilyticus CHB1 fermenting and producing CGTase crude enzyme liquid Body step is:

(1) activation of Geobacillus stearothermophilus CHB1: Geobacillus stearothermophilus CHB1 is rule with inoculating loop On bacterial strain activation medium, and in constant incubator, cultivate 18 h～24 h, and cultivation temperature is 60 DEG C～65 DEG C；

(2) seed liquor is prepared: single colony inoculation of bacstearothermophilus CHB1 step (1) obtained is in 50 In the seed culture medium of mL, and it is placed in constant temperature oscillation shaking table and cultivates 18 h～24 h, temperature 60 C～65 DEG C, rotating speed 180 rpm/min；

(3) fermentation crude enzyme liquid: by 2 % subcultivation amounts, the seed liquor that step (2) is obtained is proceeded to the product enzyme of sterilization Cultivating in culture medium, temperature 60 DEG C～65 DEG C, rotating speed 180 rpm/min, incubation time is 48 h；

(4) preparation of cyclodextrin glycosyltransferase crude enzyme liquid: the liquid fermentation liquid 10000 that step (3) is obtained G ultracentrifugation 10 min, supernatant is crude enzyme liquid；

The component of described strain activation and culture base: beef extract 0.5 %, soy peptone 0.9 %, dipotassium hydrogen phosphate 0.1 %, potassium dihydrogen phosphate 0.75 %, agar powder 1.6 %, distilled water preparation, pH 7.2；

The component of described seed culture medium: beef extract 0.5 %, soy peptone 0.9 %, dipotassium hydrogen phosphate 0.1 %, phosphorus Acid dihydride potassium 0.75 %, distilled water preparation, pH 7.2；

The component of described culture medium: dextrin 1 %, Carnis Bovis seu Bubali cream 0.3 %, soy peptone 0.5 %, CaCl₂0.2 %, K₂HPO₄ 0.2 %, KH₂PO₄ 0.2 %, MgSO₄·7H₂O, FeSO₄ 0.2 %, distilled water preparation, pH 7.0.

Beneficial effects of the present invention: the invention provides a strain stearothermophilus ground spore bacillus Geobacillus Caldoxylosilyticus CHB1, with this bacterial strain as fermentation strain, forms with dextrin, Carnis Bovis seu Bubali cream and soy peptone etc. Culture medium, fermentation obtains high temperature CGTase crude enzyme liquid.Obtained CGTase catalysis soluble starch generates alpha-cyclodextrin.This Bright provided bacterial strain has good potentiality at the producer mask of alpha-cyclodextrin.

Accompanying drawing explanation

Fig. 1 CHB1 bacterium colony figure.

Fig. 2 CHB1 morphology.

Fig. 3 CGTase product thin layer chromatography analysis.

Fig. 4 CGTase optimal reactive temperature.

Fig. 5 CGTase optimal reaction pH.

Detailed description of the invention

Embodiment 1

1, the separation of this bacterial strain

(1) 10 g soil samples are taken in 80 mL sterilizing ddH₂O shakes up, and coats NA solid plate culture medium after gradient dilution, and 60 DEG C～65 DEG C of constant incubator incubated overnight；

(2) the discrepant bacterium colony such as shape, color and size in picking step (1), solid plate line purification, pure respectively After changing three times, bacterium is protected in test tube slant；

(3) in picking step (2) purification strain dibbling in the NA solid agar flat board containing 1% soluble starch, 60 DEG C～ Overnight incubation it is inverted under 65 DEG C of constant temperature；

(4) colour developing of iodine liquid has the amylolysis named CHB1 of circle person.

The qualification of 2 bacterial strain CHB1

The growth temperature range of this bacterium is 47 DEG C～75 DEG C, and optimum growth temperature is 60 DEG C～65 DEG C；Bacterium colony is circular, yellowish Color is transparent, smooth surface, edge neat (Fig. 1)；Light microscopic hypothallus is shaft-like, acrodolichomelia born of the same parents, and brood cell is circular, and brood cell's individuality is less (Fig. 2)；This bacterium physiological and biochemical property has been carried out identifying (table 1) by application Beijing overpass company biochemical identification pipe；

These parameters combines 16S rDNA sequencing result and identifies that this bacterium is Geobacillus caldoxylosilyticus, By named for this bacterium Geobacillus caldoxylosilyticus CHB1.

Table 1 bacterial strain CHB1 physiological and biochemical property

The preparation of 3 crude enzyme liquids

(1) actication of culture: inoculating loop picking CHB1 strain lines in actication of culture solid plate culture medium, and culture medium exists 62 DEG C of constant incubator upside down incubated overnight；

(2) prepared by seed liquor: the mono-colony inoculation of CHB1 that picking step (1) obtains shakes in the triangle filling seed culture medium In Ping, shaking flask is placed in 62 DEG C of shaking tables cultivation, and seed liquid amount is 50/250 mL, and rotating speed is 180 rpm, and incubation time is 20 h；

(3) fermentation of enzyme liquid: taking seed liquor prepared by step (2) and inoculate with the inoculum concentration of 5 %, liquid amount is 40 mL/ 250 mL, cultivation temperature 60 DEG C, rotating speed is 180 rpm, and incubation time is 48 h；

(4) prepared by crude enzyme liquid: tunning 10000 g of step (3) is centrifuged 5 min, and obtained supernatant is CGTase Crude enzyme liquid.

4 fermentation enzyme liquid catalysis soluble starches produce alpha-cyclodextrin

(1) in the 1.5 mL centrifuge tubes filling 100 μ L 1% soluble starches, 100 μ L above-mentioned enzyme liquid is added, 60 DEG C React 10 min；

(2) by product in step (1) with capillary glass tube point plate in silica gel 60 thin layer chromatography board, simultaneously with α, β and γ tri- Planting cyclodextrin standard substance and carry out thin layer chromatography as comparison, developing solvent composition is: n-butyl alcohol: ethanol: water: ammonia=5:3:4: 2, iodine vapor develops the color two hours, it was demonstrated that this CGTase catalysis soluble starch principal product is alpha-cyclodextrin (Fig. 3)；

(3) take centrifugal after fermented liquid supernatant i.e. crude enzyme liquid 0.1 mL that obtains, join equipped with 0.9 ml in advance with 50 3 %(w/v that mmol/L sodium phosphate buffer (pH 6.0) is prepared) soluble starch solution test tube in, at 60 DEG C react 10 After min, the hydrochloric acid solution adding 1.0 mL 1 mol/L terminates reaction, adds 1.0 ml in advance with 50 mmol/L sodium phosphates The methyl orange solution of 0.1 mmol/L that buffer (pH 6.0) is prepared, is incubated 20 min, at wavelength 505 at 16 DEG C after mixing Absorbance is measured at nm.Calculate the concentration of alpha-cyclodextrin according to alpha-cyclodextrin standard curve, an enzyme activity unit is defined as Enzyme amount needed for the alpha-cyclodextrin of generation 1 μm ol the most per minute, the enzyme recording this crude enzyme liquid is lived as 0.5U/ ML, methyl orange method is the standard method of detection alpha-cyclodextrin, and this result also further demonstrates that the catalysis solubility of this CGTase is formed sediment The principal product of powder is alpha-cyclodextrin.

The purification of 5 CGT enzyme and character

(1) above-mentioned crude enzyme liquid through ammonium sulfate precipitation, dialyse, concentrate, after ion-exchange chromatography and polydextran gel column chromatography Obtain electrophoretically pure CGT enzyme；

(2) optimal reactive temperature of this enzyme, optimal reaction pH and Rate activity are measured with methyl orange fall color method.Concrete grammar is such as Under: after pure enzyme Bradford standard measure, dilute 10 times, 50 times, 100 times, 500 times and 1000 times respectively, take each dilution respectively Enzyme 0.1 mL of degree joins 3 %(w/v prepared in advance equipped with 0.9 ml with 50 mmol/L sodium phosphate buffers (pH 6.0)) In the test tube of soluble starch solution, after reacting 10 min at 60 DEG C, the hydrochloric acid solution adding 1.0 mL 1mol/L terminates anti- Should, add the methyl orange solution of 0.1 mmol/L that 1.0 ml prepare in advance with 50mmol/L sodium phosphate buffer (pH6.0), At 16 DEG C, it is incubated 20 min after mixing, at wavelength 505 nm, measures absorbance.Enzyme is determined according to alpha-cyclodextrin standard curve Optimum diluting multiple.The enzyme liquid utilizing said method and optimum diluting multiple measures optimal reaction pH and the optimal reaction of this enzyme Temperature, and under optimum reaction conditions, measure the Rate activity of this enzyme.Measurement result shows that the optimal reactive temperature of this CGT enzyme is 60 DEG C～65 DEG C (Fig. 4), optimal reaction pH is 5.0～6.0(Fig. 5), the Rate activity of enzyme is 112U/mg after purification

To sum up, find that the bacterial strain Geobacillus caldoxylosilyticus CHB1 of high temperature CGTase is produced in a strain, should The enzyme liquid prepared with this bacterial strain can be catalyzed soluble starch under 60 DEG C～65 DEG C of high temperature and generate alpha-cyclodextrin.

Claims (1)

1. a liquid fermentation cyclodextrin glycosyltransferase crude enzyme liquid preparation method for Geobacillus stearothermophilus bacterial strain, It is characterized in that:

Concretely comprising the following steps of its liquid fermentation crude enzyme liquid preparation method:

(1) activation of Geobacillus stearothermophilus CHB1: Geobacillus stearothermophilus CHB1 is lined bacterium with inoculating loop On strain activation medium, and in constant incubator, cultivate 18 h～24 h, and cultivation temperature is 60 DEG C～65 DEG C；

(2) seed liquor is prepared: single colony inoculation of bacstearothermophilus CHB1 step (1) obtained is in 50 mL's In seed culture medium, and it is placed in constant temperature oscillation shaking table and cultivates 18 h～24 h, temperature 60 C～65 DEG C, rotating speed 180 rpm/min；

(3) fermentation crude enzyme liquid: by 2 % subcultivation amounts, the seed liquor that step (2) is obtained is proceeded to the product enzyme cultivation of sterilization Cultivating in base, temperature 60 DEG C～65 DEG C, rotating speed 180 rpm/min, incubation time is 48 h；

(4) preparation of cyclodextrin glycosyltransferase crude enzyme liquid: liquid fermentation liquid 10000 g step (3) obtained surpasses Centrifugal 10 min of speed, supernatant is crude enzyme liquid；

Described bacterial strain is Geobacillus stearothermophilus bacterial strain (Geobacillus caldoxylosilyticus) CHB1, this bacterium Strain on August 28th, 2013 in the registration preservation of Chinese Typical Representative culture DSMZ, deposit number is CCTCC M 2013384；

The component of described strain activation and culture base: beef extract 0.5 %, soy peptone 0.9 %, dipotassium hydrogen phosphate 0.1 %, phosphorus Acid dihydride potassium 0.75 %, agar powder 1.6 %, distilled water preparation, pH 7.2；

The component of described seed culture medium: beef extract 0.5 %, soy peptone 0.9 %, dipotassium hydrogen phosphate 0.1 %, di(2-ethylhexyl)phosphate Hydrogen potassium 0.75 %, distilled water preparation, pH 7.2；

The component of described culture medium: dextrin 1 %, Carnis Bovis seu Bubali cream 0.3 %, soy peptone 0.5 %, CaCl₂0.2 %, K₂HPO₄ 0.2 %, KH₂PO₄ 0.2 %, MgSO₄·7H₂O, FeSO₄ 0.2 %, distilled water preparation, pH 7.0.