CN103205406A - Method for producing heatproof beta-amylase by using bacillus subtilis 6-7 - Google Patents
Method for producing heatproof beta-amylase by using bacillus subtilis 6-7 Download PDFInfo
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Abstract
The invention provides a method for producing heatproof beta-amylase by using wild bacillus subtilis 6-7. According to the method, a bacterium with an accession number of 6-7 is screened out from an acquired soil sample and strains preserved in a laboratory; after the bacterium is fermented in a basic medium for 64 h, enzyme activity reaches 2200.0 U/mL, and when cassava is used as a carbon source, enzyme activity reaches 5120.5 U/mL, increasing by 1.33 times; an optimal carbon and nitrogen source comprises 2% of cassava powder and 4% of soybean cake powder, optimal culture conditions comprises a temperature of 37 DEG C, a shaking speed of 190 r/min, a loading liquid volume of 50 ml in an Erlenmeyer flask with a volume of 250 ml and an inoculation amount of 4%, and enzyme activity reaches 6724.0 U/ml after shaking culture for 64 h, increasing by 2.06 times compared with enzyme activity before optimization; and the optimal reaction temperature and the optimal pH value of the beta-amylase are 65 DEG C and 6.0, respectively. The extracellular beta-amylase produced from the wild bacillus subtilis through fermentation of cassava in the invention has the advantages of heat resistance, low cost, high output and good product performance, laying a foundation for industrial production. The wild bacillus subtilis is preserved in China Center for Type Culture Collection with an accession number of CCTCC M2009200.
Description
Technical field
The present invention relates to a kind of wild-type subtilis Bacillus subtilis6-7 and produce the production bacterium of the outer heat-resisting beta-amylase of born of the same parents and optimize processing method, belong to enzyme engineering and fermentation engineering field.
Background technology
(β-amylase EC3.2.1.2) claims maltoside enzyme, glycogenase, saccharogen amylase again to beta-amylase, and its system is called α-1,4-dextran-4-maltose lytic enzyme (α-1,4-D-glucan maltohydrolase).Beta-amylase is a kind of circumscribed-type saccharifying enzyme, when acting on starch, can cut next maltose unit in turn from the non reducing end of α-1,4 glycosidic link, generate maltose and macromolecular β-limit dextrin, but can not hydrolyzing alpha-1, the 6-glycosidic link runs into the tapping point of α-1,6 key, then stagnation is so hydrolysis rate is slower.When its hydrolysis amylopectin, linear fraction generates maltose, and near the tapping point and inboard left behind because not being hydrolyzed.Owing to during this zymolyte, Walden translocation reaction (Walden inversion) takes place, make product become β-type maltose by α-type, so the name beta-amylase.
β-starch is one of main saccharifying enzyme in the starch course of processing, reaction does not need coenzyme, and mild condition, by product is few, can be used as the saccharifying agent on the industrial production such as maltose, beer, beverage, have important use to be worth in industries such as food-processing, beer fermentation, textiles, medicine, chemical industry medical analysis.Wherein microbe-derived beta-amylase is because its production cost is low, environmental hazard is little, inulinase-producing activity is high, is convenient to industrial production; Along with the continuous development of enzyme industry and people to the requirements at the higher level of maltose quality, use zymin and produce the focus that malt syrup has become research, the demand of beta-amylase constantly increases makes it become one of industrial enzyme of tool development prospect.
At present, the βDian Fenmei of selling on the market has two kinds: a kind of is microorganism (comprising bacterium and fungi) fermentative production; Another kind is that traditional technology is extracted from plant.The barley beta-amylase of plant origin is widely used in a plurality of fields always for a long time, the beta-amylase that obtains at present nearly all using by plant in industry such as happy sugar, beer.Barley beta-amylase (trade(brand)name OPTLMALT BBA) as outstanding person's energy section is most popular, but still can not satisfy the demand in the domestic production, so seek the high beta-amylase of suitability price ratio to guarantee the production needs of high malt sugar.And extract beta-amylase from plant, technology is coarse, and enzyme activity is low, has expended a large amount of biomass energies.
From delivered in 1972 also have the enzyme that presents with higher plant beta-amylase same function mechanism in the microorganism since, had been found that many microorganisms are that beta-amylase is produced bacterium, it is better than the beta-amylase of plant origin aspect heat-resisting.In recent years, the bacterial strain by the production by biological beta-amylase of report mainly contains bacillus cereus (Bacillus cereus), bacillus megaterium (Bacillus megaterium), Paenibacillus polymyxa (Bacillus polymyxa), Bacillus circulans bacillus such as (Bacillus circulans), hot anaerobic bacillus(cillus anaerobicus) genus (Thermoanaerobacterium), Rhodopseudomonas (Pseudomonas sp.), streptomyces (Streptomyces sp.) etc. both at home and abroad.The microorganism beta-amylase of now having reported mostly is middle warm type greatly, and optimal reactive temperature is 40 ℃～60 ℃; The synthetic beta-amylase optimal reactive temperature of hot sulphur clostridium (Clostridium thermosulfurog) is 75 ℃, but it is the anaerobic type bacterial strain, growth temperature is 60 ℃ of comparatively harshnesses, and be not suitable for industrial production requirement, therefore the Bacillus strain of screening product fire resistant alpha-diastase then is research emphasis.
Cassava is one of the world's three yampi classes, extensively cultivates in the torrid zone and subtropical zone, and in China torrid areas, cassava is the fifth-largest crop that is only second to paddy rice, sweet potato, sugarcane and corn, has that adaptability is strong, plantation region characteristics widely.Other cash crop utilize cassava fermentative production cost low relatively, can accomplish not strive grain with the people.Therefore exploitation is that the novel process that fermenting raw materials is produced beta-amylase has very tempting commercial promise with the cassava.
Not only temperature, pH wide accommodation and heat-resisting acid resistance are good for the outer heat-resisting beta-amylase of wild-type producing bacillus subtilis born of the same parents involved in the present invention, have showed tempting prospects for commercial application and commercial value.
Summary of the invention
Be plant at present beta-amylase main flow source, but be difficult to carry out stable supply, and turnout is limited.On the other hand, microbe-derived beta-amylase is low because of productivity, thermotolerance is relatively poor, production cost is high, seldom can realize practicability.The invention provides a kind of bacterial strain and low-cost fermentation technology thereof of production thermotolerance beta-amylase, comprise the research of screening, Optimum of culture medium, Optimizing Conditions of Fermentation and the zymologic property of bacterial strain, utilize cassava to produce the method for beta-amylase for fermenting raw materials.The invention provides the wild-type subtilis and utilize the outer beta-amylase of cassava fermentative production born of the same parents, have thermotolerance, cost is low, output is high, the advantage of good product performance, for suitability for industrialized production lays the foundation.
One of the object of the invention is the bacterial strain Bacillus subtilis6-7 that obtains a kind of production thermotolerance beta-amylase.This bacterial strain is (related application number: 201010167019.7) disclose, and submitted preservation proof and survival proof before this in applicant's patent application before this.Correlative study to this bacterial strain obtains as drawing a conclusion:
1. substratum and culture condition
LB substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, pH7.0
Starch solids substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, Zulkovsky starch 1% and agar powder 2%, pH7.0
Primary dcreening operation substratum: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, Zulkovsky starch 1%, pH7.0
Sieve substratum again: Tryptones 1%, yeast extract paste 0.5%, glucose 0.1%, Zulkovsky starch 0.5%, potassium primary phosphate 0.2%, bitter salt 0.05%, two hydration calcium chloride 0.02%, pH7.0.
Two of the object of the invention is to provide a kind of subtilis B.subtilis6-7 to produce the processing method of heat-resisting beta-amylase, and makes with low costly, and raw material sources are extensive, comprise Optimum of culture medium, Optimizing Conditions of Fermentation is achieved through the following technical solutions, and step is as follows:
(1) basic medium: 2% glucose, 2% Tryptones, 0.5% Zulkovsky starch, 1% sodium-chlor, pH7.0 draws growth curve and produces enzyme curve (as accompanying drawing 1);
(2) the C source is optimized, selecting cassava is main C source, and its optimal concentration is selected (as accompanying drawing 2);
(3) other compositions of basic medium are optimized: as organic N source, inorganic N source, inorganic salt, buffer system etc.;
(4) optimize substratum: Tapioca Starch 2%, bean cake powder 4%, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% sal epsom, 0.0111% calcium chloride, pH7.0;
(5) to fermentation culture conditions optimization: as culture temperature, initial pH, liquid amount, rotating speed, inoculum size, incubation time;
(6) shake-flask culture condition: this bacterium shakes in the bottle at the 250ml that contains 50ml optimization substratum, and initial pH is 7.0, inoculum size 4% (V/V), 160-190r/min, 37 ℃ of fermentation culture 60h.
(7) the B.subtilis6-7 bacterial strain is cultivated in basic medium, shake-flask culture 60h, and enzyme work reaches 2200.0U/ml.Through the C source, experiment of single factor such as organic N source, inorganic N source, inorganic salt, buffer system and orthogonal experiment optimize substratum, optimize culture condition again, according to optimal culture conditions shake-flask culture 60h, the highest production of enzyme reaches 6724.0U/ml in optimizing substratum, has improved 206.45% before optimizing.
Three of the object of the invention is the enzyme reaction conditions that obtain bacterial strain B.subtilis6-7 peak enzymolysis-ability starch, obtains following result:
(1) this enzyme thermal adaptation a wider range, optimum temperature is 65 ℃, and better 60～70 ℃ of temperature temperature of preserving down, and the long-time temperature stability of preservation is relatively poor more than 70 ℃, but preserve the interior remnant enzyme activity of 30min still higher (as accompanying drawing 3).
(2) this enzyme optimal reaction pH value is 6.0, and high enzyme vigor (as accompanying drawing 4) is all arranged between pH5-7.
(3) this enzyme molecular weight is about 65KD.
(4) effect: act on α-1,4 glycosidic link of polyose and oligosaccharides class, maltose is free;
(5) effect substrate specificity: hydrolysis Zulkovsky starch, amylose starch, amylopectin, glycogen etc., can not the hydrolysis beta-cyclodextrin, trisaccharide maltose.
Beneficial effect of the present invention: the present invention passes through the subtilis Bacillus subtilis6-7 of the heat-resisting acidproof beta-amylase of screening acquisition high yield from the soil sample of the bacterial strain in laboratory preservation and collection, behind fermentation optimization, acquisition is that carbon source and bean cake powder are the method that nitrogen source fermentation is produced beta-amylase with the Tapioca Starch of cheapness and wide material sources, greatly reduces production cost.
Description of drawings
Growth and the product enzyme curve of Figure 1B .subtilis6-7 in basic medium
Fig. 2 is different, and carbon source is produced the enzyme effect to B.subtilis6-7
The beta-amylase vigor of B.subtilis6-7 under Fig. 3 differential responses temperature
The beta-amylase vigor of B.subtilis6-7 under Fig. 4 differential responses pH
Embodiment
Below, the present invention will be further detailed with embodiment, but it is not limited to these embodiment any.
Embodiment 1: the screening of producing amylase strain
The separation of bacillus purifying: 25 parts of soil samples of getting collection are made soil supension through 80 ℃ of thermal treatment 15min, 37 ℃ of enrichment culture 24h, cultivate 24h for 37 ℃ in the gradient dilution coating LB solid plate, single bacterium colony is isolated in line again, purebred until obtaining, and with purebred strain number and slant preservation, the preservation of glycerine frozen pipe.Spore staining then, gram stain microscopy determine that the bacterium colony of screening is genus bacillus.Bacterial classification with the laboratory preservation reactivates simultaneously, and thermal treatment is coated on the LB solid plate again, filters out genus bacillus, slant preservation, the preservation of glycerine frozen pipe and numbering
Produce amylase strain primary election: the genus bacillus dibbling that separation and purification is obtained is cultivated 24h for 37 ℃, 0.05% iodine staining bacterium colony, the diameter of observing transparent circle and bacterium colony circle, and record in the starch solids substratum.The colony inoculation that hydrolysis loop diameter (D)/colony diameter (d) ratio is big is cultivated 12h in the LB substratum, the glycerine frozen pipe is preserved in-40 ℃.
Primary dcreening operation: the colony inoculation that hydrolysis loop diameter and colony diameter ratio is big activates in the LB substratum, transfers in the primary dcreening operation substratum, cultivates 24h in 37 ℃, 160rpm, measures fermentation broth enzyme and lives.
Multiple sieve: the primary dcreening operation enzyme is lived higher bacterial strain through the activation of LB substratum, transfer in sieving again in the substratum, cultivate 24h in 37 ℃, 160rpm, it is alive to measure fermentation broth enzyme.Determine enzyme the highest bacterial strain called after 6-7 alive, as the research starting strain.
Embodiment 2: the mensuration of amylase activity
The mensuration that the amylase enzyme is lived adopts the Bernfeld improved method, and namely the DNS measuring method utilizes beta-amylase and Zulkovsky starch effect, discharge the reductibility of maltose residue, 3,5-dinitrosalicylic acid is reduced into 3-amino-5-nitrosalicylic acid, uses the content of spectrophotometric determination reducing sugar again.In the 25ml colorimetric cylinder, add 1ml2% Zulkovsky starch solution, 1ml pH6.0 citrate buffer solution in 40 ℃ of water-bath 5min, adds the crude enzyme liquid 1ml of dilution certain multiple, mixing, in 40 ℃ of water-bath 30min reactions, reaction finishes the back and adds 2ml3,5-edlefsen's reagent, boil 10ml in the boiling water, the flowing water cooling, deionized water is settled to 25ml, measures light absorption value under 540nm.
The live definition of unit of enzyme: under 70 ℃, the above-mentioned reaction conditions of pH6.0, it is 1U that 1ml liquid enzymes 30min hydrolyzed starch produces the required enzyme amount of reducing sugar that is equivalent to 1 μ mol maltose, represents with U/ml.
The optimization of embodiment 3 fermention mediums and culture condition
(1) drafting of growth curve and product enzyme curve: (2% glucose, 2% Tryptones, 0.5% Zulkovsky starch, 1% sodium-chlor pH7.0) are measured the growth curve of 6-7 bacterial strain in basic medium, as shown in Figure 1 at basic medium.This bacterium is cultivated after 12 hours under the condition of 160r/min (rotary shaking table) and enters logarithmic phase at 37 ℃, and 40h enters the stationary phase of growing, and beta-amylase reaches the highest enzyme 2200.0U/mL alive in 60h accumulative total.Synthetic and thalli growth half coupled mode of beta-amylase that hence one can see that.
(2) optimization in C source: change the glucose in the basic medium into Zulkovsky starch, ground rice, Semen Maydis powder, Tapioca Starch, molasses, chitosan, maltose, alpha-lactose, glucose, beta-cyclodextrin, plant 12h in age, 4%, 37 ℃ of inoculum size, 160r/min fermentation culture.Cassava is that the enzyme activity in C source can reach 5120.5U/mL, is that Zulkovsky starch is 2.55 times of C source, is that glucose is 2.33 times of C source, is that Semen Maydis powder is 2.03 times of C source, is that ground rice is 1.87 times of C source, illustrates that namely cassava is with the obvious advantage for the fermentation of C source
(2) medium optimization: change the N source of basic medium into other organic N source, determine optimum N source and optimal concentration thereof; Add different inorganic salt, determine to promote to produce the inorganic salt of enzyme; Add different inorganic nitrogens, determine the suitableeest inorganic nitrogen and add concentration; Add different buffer systems, determine the optimized buffer system; According to experiment of single factor, determine the factor that influence is big, design 4 factors, 3 water-glasses, carry out orthogonal optimization, obtain the optimal medium combination.
(3) optimization of fermentation culture conditions: in optimizing substratum, carry out the optimization of culture condition, as culture temperature, rotating speed, initial pH, liquid amount, inoculum size etc., determine that optimal culture condition is: 37 ℃, 190r/min, initial pH7.0,50mL liquid amount are in 250mL triangular flask, 4% inoculum size
(4) enzyme activity determination: this bacterium shakes at the 250ml that contains 50ml optimization substratum and carries out shake flask fermentation in the bottle, 4%, 37 ℃ of inoculum size, 190r/min cultivate 60h, centrifuging and taking fermented supernatant fluid, record enzymic activity and reach 6724.0U/ml, improved 2.05 times than 2200.0U/mL before optimizing.
Embodiment 4: heat-resisting beta-amylase zymologic property
(1) temperature is to the influence of enzymic activity
The same reaction system is measured this amylase enzyme activity respectively under 30～80 ℃, the result as shown in Figure 2: this enzyme all shows higher enzymic activity between 55～75 ℃, illustrate that the temperature optimum range of this enzyme is wideer, and its optimum temperature is 65 ℃.
(2) influence of the enzymic activity of pH
The same reaction system is surveyed this enzyme enzyme respectively and is lived under 3-8.0, the result as shown in Figure 3, this enzyme enzymic activity when pH5-7 is higher, the suitableeest action pH is 6.0.
(3) mensuration of amylase protein molecular weight size
The crude enzyme liquid that fermentation is obtained passes through SDS-PAGE analyzing proteins molecular weight, and this enzyme molecular weight is about 65KD.
Claims (5)
1. a bacillus subtilis Bacillus subtilis6-7 has been preserved in Chinese typical culture collection center, and deposit number is: CTCC M2009200.
2. subtilis as claimed in claim 1 is characterized in that, this bacterium is the safe bacterial strain of wild-type, and the outer beta-amylase of the born of the same parents of production is that 65 ℃ of enzyme activities are the highest in temperature of reaction, has very high thermotolerance.
3. the beta-amylase produced of subtilis as claimed in claim 1 is characterized in that the optimal reaction pH of this enzyme is 6.0, at pH is to use under the condition of 4.5-7.5.
4. the beta-amylase produced of the described subtilis of claim 1 has following characteristic:
(1) this enzyme molecular weight is about 66KD;
(2) effect: act on α-1,4 glycosidic link of polyose and oligosaccharides class, generate free beta-maltose;
(3) effect substrate specificity: hydrolysis Zulkovsky starch, amylose starch, amylopectin, glycogen etc., can not the hydrolysis beta-cyclodextrin, trisaccharide maltose;
(4) hydrolysis Zulkovsky starch product is mainly maltose, is a spot of trisaccharide maltose, glucose secondly, does not have oligose such as maltotetrose, maltopentaose, MALTOHAXAOASE.
5. the heat-resisting subtilis 6-7 of high yield as claimed in claim 1, its fermentation culture method:
(1) seed culture: seed culture medium is 1% peptone, 0.5% yeast extract paste, 0.5% Zulkovsky starch, 1% sodium-chlor, pH7.0, the bacterial strain of-40 ℃ of glycerine frozen pipes preservation is inserted in the seed culture medium, shake a bottle shaking culture 12h at 37 ℃ and 160r/min, be forwarded to again and cultivate 12h in the fresh seed culture medium as secondary seed solution;
(2) carbon source cassava: Tapioca Starch is main carbon source material source, and content is 2%～4%, and the cassava piece is directly broken into pieces, and mechanical grinding becomes powder, and 40 orders sieve, and directly takes by weighing Tapioca Starch during the substratum preparation and puts into triangular flask, handles without enzymolysis or acidolysis;
(3) optimize other compositions of substratum: bean cake powder 4%, 0.1% Secondary ammonium phosphate, 0.00139% ferrous sulfate, 0.6% Trisodium Citrate, 0.4% potassium primary phosphate, 0.0005% zinc sulfate, 0.0123% sal epsom, 0.0111% calcium chloride, pH7.0, prepare with deionized water;
(4) sterilization: 121 ℃ of sterilization 25min;
(5) conditions of flask fermentation: the fermention medium of the bottled 50ml of 250ml triangle, inoculum size are 4% (volume ratio), 37 ℃ and 190r/min shaking table shaking culture, and fermentation 64h, enzyme work reaches 6724U/ml.
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| CN110144312A (en) * | 2019-05-24 | 2019-08-20 | 江南大学 | It is a kind of produce maltotetraose forming amylase bacillus alcalophilus and its application |
| CN114644984A (en) * | 2022-04-28 | 2022-06-21 | 临沂大学 | Method for separating microorganism producing amylase from soil |
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| CN101851598A (en) * | 2010-05-10 | 2010-10-06 | 江南大学 | Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate |
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| CN110144312A (en) * | 2019-05-24 | 2019-08-20 | 江南大学 | It is a kind of produce maltotetraose forming amylase bacillus alcalophilus and its application |
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| CN114644984A (en) * | 2022-04-28 | 2022-06-21 | 临沂大学 | Method for separating microorganism producing amylase from soil |
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