CN103981125A - Geobacillus caldoxylosilyticus strain producing cyclodextrin glycosyltransferase - Google Patents

Abstract

The present invention relates to a Geobacillus caldoxylosilyticus strain producing cyclodextrin glycosyltransferase, wherein the strain is Geobacillus caldoxylosilyticus CHB1, is preserved in China Center for Type Culture Collection on August 28, 2013, and has the preservation number of CCTCC M2013384, and it is proved that the enzyme produced by the Geobacillus caldoxylosilyticus is cyclodextrin glycosyltransferase (short for CGTase) belonging to the alpha-amylase family, and can be adopted to catalyze soluble starch so as to generate alpha-cyclodextrin. According to the present invention, the new strain Geobacillussp CHB1 producing CGTase is provided, the optimum enzyme activity reaction temperature of the enzyme production fermentation broth supernatant of the strain is 65 DEG C, the activity is maintained between 40-85 DEG C, and the CGTase produced by the strain can be used for producing alpha-cyclodextrin, and can further be used for processing processes of starch in the temperature range and the like.

Description

The stearothermophilus ground bacillus bacterial strain of cyclomaltodextrin glucanotransferase is produced in one strain

Technical field

The present invention relates to a kind of stearothermophilus ground bacillus and application thereof, especially find a kind of new product high temperature cyclodextrin glucosyltransferase (CGTase) bacterial strain Geobacillus sp and apply this strain fermentation and produce cyclodextrin glucosyltransferase, belonging to fermentative production field.

Background technology

Cyclomaltodextrin glucanotransferase is a kind of starch enzyme that can catalysis starch changes into cyclodextrine, belongs to the important member of alpha-amylase family, and it has cyclisation, disproportionation, coupling and four kinds of activity of hydrolysis.In production, main its cyclization of application generates cyclodextrin, and cyclodextrin glucosyltransferase catalysate is α, β and tri-kinds of cyclodextrines of γ, is joined end to end and forms respectively by 6,7 and 8 glucose glycan molecules with α-1.4 glycosidic link.With principal product difference, define cyclodextrin glucosyltransferase and can be divided into again alpha-cylodextrin Transglucosylase, beta-cyclodextrin Transglucosylase and γ-cyclodextrin Transglucosylase.The multi-effects such as the cavity structure of the outside hydrophilic inner hydrophobic of cyclodextrin can play slowly-releasing in conjunction with multiple small-molecule substance, covers smell, protection, have important application at medicine, food, makeup and the field such as agriculture.The production of cyclodextrin is mainly that application CGTase catalysis obtains, and the CGTase of domestic application and report produces bacterial strain and mostly is the normal temperature bacterium such as Bacillus genuss at present, the CGTase that produces be also normal temperature enzyme.High temperature enzyme has advantages of that the normal temperature enzymes such as catalytic rate is fast, tolerable temperature is high are incomparable.The inventor finds the bacterial strain of a strain product high temperature CGTase, and there is no at present the report that other bacterial strain of the same race of Geobacillus caldoxylosilyticus produces this enzyme, by liquid fermenting, obtain crude enzyme liquid, this crude enzyme liquid can produce cyclodextrin for efficient catalytic Zulkovsky starch.Based on above discovery, the present invention has been proposed.

summary of the invention

The object of this invention is to provide a strain and produce the stearothermophilus ground bacillus bacterial strain of cyclomaltodextrin glucanotransferase, this bacterial strain is stearothermophilus ground bacillus CHB1, be accredited as Geobacillus caldoxylosilyticus, called after Geobacillus caldoxylosilyticu CHB1, this bacterial strain is on August 28th, 2013 in the registration preservation of Chinese Typical Representative culture DSMZ, and deposit number is CCTCC M 2013384.And with this strain fermentation, generate CGTase crude enzyme liquid, apply this enzyme liquid and can generate principal product alpha-cylodextrin at high temperature (40 ℃～85 ℃) condition catalysis Zulkovsky starch, optimal reactive temperature is 60 ℃～65 ℃.

The present invention implements by the following technical programs:

First laboratory is separated to the bacterial strain CHB1 that dextrin glucanotransferase is produced in a strain from the soil of Foochow one hen house compost, after identifying, concrete Classification And Nomenclature is Geobacillus caldoxylosilyticus CHB1, culture presevation is registered preservation on August 28th, 2013 in Chinese Typical Representative culture DSMZ, deposit number is CCTCC M 2013384, and preservation address is Wuhan University.

By the concrete steps of described bacterial strain Geobacillus caldoxylosilyticus CHB1 fermentative production CGTase crude enzyme liquid, be:

(1) activation of stearothermophilus ground bacillus CHB1: with transfering loop, stearothermophilus ground bacillus CHB1 is lined on bacterial strain activation medium, and cultivate 18 h～24 h in constant incubator, and culture temperature is 60 ℃～65 ℃;

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

(3) fermentation crude enzyme liquid: seed liquor step (2) being obtained by 2 % culture transferring amounts proceeds in the culture medium of sterilization and cultivates, 60 ℃～65 ℃ of temperature, rotating speed 180 rpm/min, incubation time is 48 h;

(4) preparation of cyclomaltodextrin glucanotransferase crude enzyme liquid: liquid fermentation liquid 10000 g ultracentrifugation 10 min that step (3) is obtained, 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 primary 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 %, potassium primary phosphate 0.75 %, distilled water preparation, pH 7.2;

The component of described culture medium: dextrin 1 %, extractum carnis 0.3 %, soy peptone 0.5 %, CaCl ₂0.2 %, K ₂hPO ₄0.2 %, KH ₂pO ₄0.2 %, MgSO ₄7H ₂o, FeSO ₄0.2 %, distilled water preparation, pH 7.0.

Beneficial effect of the present invention: the invention provides a strain stearothermophilus ground gemma rod-shaped bacterium Geobacillus caldoxylosilyticus CHB1, with this bacterial strain as fermentation strain, with dextrin, extractum carnis and soy peptone etc., form culture medium, fermentation obtains high temperature CGTase crude enzyme liquid.Institute's CGTase catalysis Zulkovsky starch that obtains generates alpha-cylodextrin.Bacterial strain provided by the present invention has good potentiality at the producer mask of alpha-cylodextrin.

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.

Embodiment

Embodiment 1

1, the separation of this bacterial strain

(1) get 10 g soil samples in 80 mL sterilizing ddH ₂o shakes up, and coats NA solid plate substratum after gradient dilution, 60 ℃～65 ℃ constant incubator incubated overnight;

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

(3) in picking step (2), the dibbling of purifying bacterial classification, in the NA solid agar plate containing 1% Zulkovsky starch, is inverted overnight incubation under 60 ℃～65 ℃ constant temperature;

(4) colour developing of iodine liquid has amylolysis circle person called after CHB1.

The evaluation of 2 bacterial strain CHB1

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

These parameters identifies that in conjunction with 16S rDNA sequencing result this bacterium is Geobacillus caldoxylosilyticus, by this bacterium called after Geobacillus caldoxylosilyticus CHB1.

Table 1 bacterial strain CHB1 physiological and biochemical property

The preparation of 3 crude enzyme liquids

(1) actication of culture: transfering loop picking CHB1 bacterial classification lines on actication of culture solid plate substratum, substratum is 62 ℃ of constant incubator upside down incubated overnight;

(2) seed liquor preparation: the mono-colony inoculation of CHB1 that picking step (1) obtains is in filling the triangle shaking flask of seed culture medium, and shaking flask is placed in 62 ℃ of shaking tables and cultivates, 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: get seed liquor prepared by step (2) with the inoculum size inoculation of 5 %, liquid amount is 40 mL/250 mL, 60 ℃ of culture temperature, rotating speed is 180 rpm, incubation time is 48 h;

(4) crude enzyme liquid preparation: by centrifugal 5 min of tunning 10000 g of step (3), the supernatant that obtains is CGTase crude enzyme liquid.

4 fermenting enzyme liquid catalysis Zulkovsky starches produce alpha-cylodextrin

(1) to filling in 1.5 mL centrifuge tubes of 100 μ L 1% Zulkovsky starches, add the above-mentioned enzyme liquid of 100 μ L, 60 ℃ of reaction 10 min;

(2) by product in step (1) with glass capillary point plate in silica gel 60 thin layer chromatography board, simultaneously with α, tri-kinds of cyclodextrin standard substance of β and γ carry out thin layer chromatography in contrast, developping agent composition is: propyl carbinol: ethanol: water: ammoniacal liquor=5:3:4:2, iodine vapor colour developing two hours, proves that this CGTase catalysis Zulkovsky starch principal product is alpha-cylodextrin (Fig. 3);

(3) getting the fermented liquid supernatant obtaining after centrifugal is crude enzyme liquid 0.1 mL, join 3 %(w/v that 0.9 ml uses 50 mmol/L sodium phosphate buffers (pH 6.0) preparations be in advance housed) in the test tube of Zulkovsky starch solution, at 60 ℃, react after 10 min, the hydrochloric acid soln termination reaction that adds 1.0 mL 1 mol/L, add again 1.0 ml to use in advance the methyl orange solution of 0.1 mmol/L of 50 mmol/L sodium phosphate buffers (pH 6.0) preparation, after mixing, at 16 ℃, be incubated 20 min, at wavelength, 505 nm places measure absorbancy.According to alpha-cylodextrin typical curve, calculate the concentration of alpha-cylodextrin, enzyme activity unit is defined as the required enzyme amount of alpha-cylodextrin that under above-mentioned reaction conditions per minute generates 1 μ mol, the enzyme that records this crude enzyme liquid is lived as 0.5U/mL, tropeolin-D method is to detect the standard method of alpha-cylodextrin, and this result also further proves that the principal product of the catalysis Zulkovsky starch of this CGTase is alpha-cylodextrin.

The Purification and properties of 5 CGT enzymes

(1) above-mentioned crude enzyme liquid obtains electrophoretically pure CGT enzyme after ammonium sulfate precipitation, dialysis, concentrated, ion exchange chromatography and dextrane gel column chromatography;

(2) by tropeolin-D, falling color method measures the optimal reactive temperature of this enzyme, optimal reaction pH and compares vigor.Concrete grammar is as follows: by pure enzyme with diluting respectively 10 times after Bradford standard measure, 50 times, 100 times, 500 times and 1000 times, get respectively each dilution enzyme 0.1 mL join 3 %(w/v that 0.9 ml uses 50 mmol/L sodium phosphate buffers (pH 6.0) preparations are in advance housed) in the test tube of Zulkovsky starch solution, at 60 ℃, react after 10 min, the hydrochloric acid soln termination reaction that adds 1.0 mL 1mol/L, add again 1.0 ml to use in advance the methyl orange solution of 0.1 mmol/L of 50mmol/L sodium phosphate buffer (pH6.0) preparation, after mixing, at 16 ℃, be incubated 20 min, at wavelength, 505 nm places measure absorbancy.According to alpha-cylodextrin typical curve, determine the optimum diluting multiple of enzyme.Utilize the enzyme liquid of aforesaid method and optimum diluting multiple to measure optimal reaction pH and the optimal reactive temperature of this enzyme, and under optimum reaction conditions, measure the ratio vigor of this enzyme.Measurement result shows that the optimal reactive temperature of this CGT enzyme is 60 ℃～65 ℃ (Fig. 4), and optimal reaction pH is 5.0～6.0(Fig. 5), after purifying, the ratio vigor of enzyme is 112U/mg

To sum up, find that a strain produces the bacterial strain Geobacillus caldoxylosilyticus CHB1 of high temperature CGTase, apply enzyme liquid prepared by this bacterial strain and can under 60 ℃～65 ℃ high temperature, generate alpha-cylodextrin for catalysis Zulkovsky starch.

  

Claims (3)

1. stearothermophilus ground bacterial strain of bacillus, it is characterized in that: described bacterial strain is stearothermophilus ground bacterial strain of bacillus (Geobacillus caldoxylosilyticus) CHB1, this bacterial strain is registered preservation on August 28th, 2013 in Chinese Typical Representative culture DSMZ, and deposit number is CCTCC M 2013384.

2. the preparation method that a kind of stearothermophilus ground bacterial strain of bacillus is produced cyclodextrin glucosyltransferase as claimed in claim 1, is characterized in that: its liquid fermenting crude enzyme liquid preparation method's concrete steps are:

(1) activation of stearothermophilus ground bacillus CHB1: with transfering loop, stearothermophilus ground bacillus CHB1 is lined on bacterial strain activation medium, and cultivate 18 h～24 h in constant incubator, and culture temperature is 60 ℃～65 ℃;

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

(3) fermentation crude enzyme liquid: seed liquor step (2) being obtained by 2 % culture transferring amounts proceeds in the culture medium of sterilization and cultivates, 60 ℃～65 ℃ of temperature, rotating speed 180 rpm/min, incubation time is 48 h;

(4) preparation of cyclomaltodextrin glucanotransferase crude enzyme liquid: liquid fermentation liquid 10000 g ultracentrifugation 10 min that step (3) is obtained, supernatant is crude enzyme liquid.

3. the preparation method that a kind of stearothermophilus ground bacterial strain of bacillus is produced cyclodextrin glucosyltransferase according to claim 2, it is characterized in that: the component of described strain activation and culture base: beef extract 0.5 %, soy peptone 0.9 %, dipotassium hydrogen phosphate 0.1 %, potassium primary 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 %, potassium primary phosphate 0.75 %, distilled water preparation, pH 7.2;

The component of described culture medium: dextrin 1 %, extractum carnis 0.3 %, soy peptone 0.5 %, CaCl ₂0.2 %, K ₂hPO ₄0.2 %, KH ₂pO ₄0.2 %, MgSO ₄7H ₂o, FeSO ₄0.2 %, distilled water preparation, pH 7.0.