CN104630084A - Bacillus subtilis producing high-temperature-resistance alpha-amylase - Google Patents
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Abstract
The invention discloses bacillus subtilis producing high-temperature-resistance alpha-amylase. The bacillus subtilis Li-2013-02 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 15, 2013, and has a preservation number of CGMCC No.7926. The strain is prepared from bacillus subtilis Li-2010 producing high-temperature-resistance alpha-amylase by means of ultraviolet ray-lithium chloride-diethyl sulfate combined mutation screening, has the characteristics of strong acid resistance, heat resistance and high enzyme production activity, and has a wide application prospect.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the subtilis that Thermostable α-Amylase is produced in a strain.
Background technology
α-amylase full name is α-Isosorbide-5-Nitrae-glucan hydrolase (EC3.2.1.1), when acting on starch, α-1 can be cut from intramolecule, 4-glycosidic link and generate dextrin and reducing sugar, because the terminal glucose saccharide residue C1 carbon atom of product is α-configuration, therefore must be called α-amylase.
Thermostable α-Amylase is extensively present in animal (saliva, pancreas), plant (barley) and microorganism, is a kind of Endoglucanases.It can α-1 at a higher temperature rapidly in hydrolyzed starch molecule, 4 glucoside bonds, any cutting starch becomes short chain dextrin different in size and a small amount of oligose, glucose and maltose, thus the viscosity of starch paste is declined rapidly, be one of current most important industrial enzyme preparation, account for 25% of the zymin market share.Thermostable α-Amylase is different from mesophilicα-diastase and common high-temperatureα-amylase, there is the characteristics such as the high and wider pH subject range of superior resistance toheat, enzyme activity, be widely used in the industrial circles such as papermaking, weaving, printing and dyeing, citric acid, lactic fermentation, beer, alcohol, monosodium glutamate, β-amylose (glucose, maltose, dextrin, fructose, oligose), consumption is large, share of market is high, and application prospect is boundless.
The source of Thermostable α-Amylase is mainly ancient bacterium and eubacterium, optimum temperature is all more than 60 DEG C, ancient bacterium generally survives under various extreme condition, as high temperature, height ooze, strong acid, highly basic etc., a lot of protein maintaining their vital movement are also adapt to its living environment.Therefore, the Thermostable α-Amylase in ancient bacterium source is subject to higher temperature, although ancient bacterium α-amylase resistance toheat is better compared with eubacterium ability, but their optimum growth temperatures are higher, mostly be more than 80 DEG C, growth conditions is comparatively harsh, and is not suitable for the requirement of suitability for industrialized production.Therefore, bacillus remains the emphasis of research.From the fifties, people just set about from high temperature soil, screening starting strain to study Thermostable α-Amylase.People are in high temperature soil in recent years, particularly near hot spring in soil, screened the Bacillus coagulans of Thermostable α-Amylase, bacstearothermophilus, bacillus acidocldarius, visible high temperature bacterium is still very important in Thermostable α-Amylase research.From Madsen in 1973 etc. report and adopt normal temperature Bacillus licheniformis to obtain a kind of new Thermostable α-Amylase, people attempt from mesophilic bacteria, as obtained Thermostable α-Amylase in subtilis, Bacillus licheniformis, this opens a new way to the research of Thermostable α-Amylase.Mostly the Thermostable α-Amylase of current industrial applications is from normal temperature bacillus.
Subtilis is current industrial enzyme preparation production application one of bacterial classification the most widely.Because subtilis habitat is various, available nutritive substance kind is very abundant, which dictates that and himself contains abundant product enzyme system, possesses the application potential producing multiple enzyme.Research data shows, subtilis can produce tens kinds of enzymes such as proteolytic enzyme, α-amylase, cellulase, beta-glucanase, phytase, polygalacturonase and zytase.According to incompletely statistics, the enzyme that subtilis produces accounts for 50% of whole enzyme market.And yield of enzyme is high, kind is many, security is good and environmental protection, be widely used as producing bacterial classification in modern industry is produced.
Chinese patent CN102363761A discloses a kind of optimization method producing high-temperature-resistant alpha-amylase strains, Bacillus licheniformis 10181 is adopted to be optimized from actication of culture, the bacterial classification whole process of fermented extracted that spreads cultivation, its enzyme of the Thermostable α-Amylase that this bacterial classification produces is lived general at 2500u/ml, and tolerable temperature is 95 DEG C.Chinese patent CN101838635A discloses a kind of preparation method of fire resistant alpha-diastase, Bacillus licheniformis is carried out rejuvenation of spawn cultivation, seed culture, through its enzyme activity of Thermostable α-Amylase of liquid submerged fermentation up to 20000-22000u/ml, tolerable temperature is up to 110 DEG C.Chinese patent CN100415879C discloses acid-proof and high-temperature resistant alpha-amylase and preparation method thereof, by recombinant DNA technology rite-directed mutagenesis precursor α-amylase, precursor alpha-amylase gene is separated from microorganism particularly Bacillus licheniformis, the amino-acid residue of itself L134 and S320 is suddenlyd change, and intestinal bacteria, subtilis, high expression alpha-amylase mutant in the bacteriums such as Bacillus licheniformis, comparatively be suitable for expressing in subtilis, make acidproof pH4.0-4.5, and the α-amylase of high temperature 70-90 DEG C is able to high expression. alpha-amylase mutant has good acid acceptance, be more suitable for industrial applications, for acid resistance, the industrialized production of Thermostable α-Amylase provides a feasible way.
As mentioned above, along with the development of biotechnology, have the production that more isolate, mutagenic bacteria, genetic engineering bacterium and combination thereof can be applied to Thermostable α-Amylase, to meet growing industrial production demand.
At present, although there are a lot of those skilled in the art to carry out unremitting research to production Thermostable α-Amylase, but still have the following disadvantages: 1. enzyme activity is low, and postprocessing working procedures is complicated, production cost is relatively high; 2. the temperature of tolerance is difficult to more than 95 DEG C, and liquefaction effect is undesirable; 3. optimum pH scope is narrower, is not suitable for " two enzyme process " production technique; 4. fermentation and leaching process contain impurity, and range of application is restricted.In order to reduce production cost, expand action condition and scope, meet the need of market, enzyme activity is high, bacterial strain that action condition is wide in range to need exploitation or optimization one strain to produce, and directly filters out to have that production stability is good, bacterial classification adapts to the initial step that strong starting strain is a lot of research.
Summary of the invention
The object of this invention is to provide the subtilis that Thermostable α-Amylase is produced in a strain.
The bacterial strain of product Thermostable α-Amylase provided by the invention is specially subtilis (Bacillus subtilis) Li-2013-02.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute) on July 15th, 2013, and preserving number is CGMCCNo.7926.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into the positive.
Described subtilis (Bacillussubtilis) Li-2013-02 is preserved in the product Thermostable α-Amylase in this laboratory subtilis Li-2010 by a strain obtains through UV-LiCl-ethyl sulfate Mutation screening, and concrete screening step is as follows:
(1) preparation of bacteria suspension
The mono-bacterium colony of Li-2010 grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 40 DEG C of cultivation 12h, after getting 1mL medium centrifugal, use brine twice, and resuspended with 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.Bacterium liquid through irradiating is coated lithium chloride flat board after gradient dilution, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 40 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle choose preserve to inclined-plane with colony diameter ratio the maximum, bacteria suspension is mixed with after purifying, fully mix with ethyl sulfate stoste after gradient dilution, and in 40 DEG C of oscillation treatment 40min, the bacterium liquid processed is coated lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 40 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium Li-2013-A, Li-2013-02, Li-2013-B after purifying
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-A obtained, Li-2013-02, Li-2013-B carry out shake flask fermentation in the 250mL shaking flask containing 30mL fermention medium, seed inoculum size 10% (V/V), 40 DEG C, 100r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid.
(5) enzyme activity determination
The definition of Mei Huo unit: 1mL crude enzyme liquid, in 105 DEG C, under pH4.2 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain Li-2013-02 is stable most superior strain, and enzyme is lived and reached 31500U/mL.
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%.
Described seed culture medium: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Fermented by bacterial strain K1-1 and obtain a kind of high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is between 101-110 DEG C, and the temperature stability of preserving below 110 DEG C is better, and more than 110 DEG C to preserve long-time temperature stability poor.
(2) this enzyme optimal reaction pH value is 4-5.High enzyme vigor is all had, the enzyme complete stability alive when pH value is 3.0 between pH value 3.0-7.0.
(3) enzymic activity: by mutant strain Li-2013-02 provided by the present invention, the Thermostable α-Amylase enzyme activity of preparation is 30000-35000U/ml.
Beneficial effect:
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the subtilis Li-2013-02 of a plant height product Thermostable α-Amylase, and this bacterial strain has acidproof, thermotolerance by force, produces the feature that enzyme activity is high.
2, this bacterial strain is had to produce the Thermostable α-Amylase enzyme activity of gained up to 30000-35000u/ml; Applicable temperature scope is 101-110 DEG C, optimal reactive temperature 103 DEG C, at 108 DEG C of enzymes complete stability alive; Being suitable for pH value in reaction scope is 3.0-7.0, the enzyme complete stability alive when pH value is 3.0, optimal reaction pH value is 4-5, higher than existing Thermostable α-Amylase enzyme activity, enzyme effect optimum pH wide scope, resistance to temperature is high, is particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1: UV-LiCl Mutation screening
(1) preparation of bacteria suspension
The mono-bacterium colony of Li-2010 grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 40 DEG C of cultivation 12h, after getting 1mL medium centrifugal, use brine twice, and resuspended with 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
3mL bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.Bacterium liquid (10-1-10-5) after gradient dilution through irradiating is coated lithium chloride flat board, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 40 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle choose preserve to inclined-plane with colony diameter ratio the maximum, bacteria suspension is mixed with after purifying, after gradient dilution, (10-1-10-5) gets 20mL bacteria suspension and fully mixes with 1mL ethyl sulfate stoste, and in 40 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 40 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium Li-2013-A, Li-2013-02, Li-2013-B after purifying;
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-A obtained, Li-2013-02, Li-2013-B carries out shake flask fermentation in the 250mL shaking flask containing 30mL fermention medium, seed inoculum size 10% (V/V), 40 DEG C, 100r/min cultivates 72h, get the centrifugal 10min of fermented liquid 5000r/min and remove thalline, supernatant liquor carries out ultrafiltration and concentration through 10000MWCO, obtain crude enzyme liquid, the enzyme measuring the crude enzyme liquid of above-mentioned three strain bacterial strains is respectively lived.
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH) 2SO40.4%, K2HPO40.8%, CaCl20.2%, lithium chloride 0.9%, agar 2%.
Described seed culture medium: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Embodiment 2: the mensuration of amylase activity
(1) definition of Mei Huo unit: 1mL crude enzyme liquid, in 110 DEG C, under pH4.5 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
(2) adopt DNS method to measure the amylase activity of Li-2010, Li-2013-A, Li-2013-02, Li-2013-B, learn, Li-2013-02 has stable most enzymatic activity high, reaches 31500U/mL.
Embodiment 3: the mensuration of the mensuration zymology of zymologic property
(1) impact of temperature on enzymic activity and the thermostability of enzyme
Same reaction system, measures this amylase enzyme activity respectively at 80 ~ 110 DEG C, and result shows that this enzyme all shows high enzyme activity between 90-110 DEG C, and thermal adaptation a wider range of this enzyme is described, its optimum temperature is 100-110 DEG C.
(2) pH is on the impact of enzymic activity and acid acceptance research
Same reaction system, under pH3-8.5, survey this enzyme enzyme respectively live, result shows that this enzyme enzymic activity when pH3-7 is higher, shows that this enzyme is acid starch enzyme.The stability of this enzyme under different pH value: the Sodium phosphate dibasic-citrate buffer solution of enzyme by different pH value is suitably diluted, survey this enzyme enzyme be incubated 18h at 25 DEG C after again to live, result shows: at pH5-8, all there is higher remnant enzyme activity, but pH5-7 is more stable, still have the enzyme of more than 75% to live after 18h room temperature preservation, illustrate that the resistance to pH value range of this enzyme more extensively and more stable in acid condition.
Claims (3)
1. the subtilis (Bacillus subtilis) of Thermostable α-Amylase is produced in a strain, and described subtilis (Bacillus subtilis) deposit number is CGMCCNo.7926.
2. the subtilis (Bacillus subtilis) of Thermostable α-Amylase is produced in a strain as claimed in claim 1, and it is characterized in that, described subtilis can produce Thermostable α-Amylase.
3. subtilis (Bacillus subtilis) Li-2013-02 that Thermostable α-Amylase is produced in a strain as claimed in claim 1 is characterized in that, described subtilis is obtained through UV-LiCl-ethyl sulfate Mutation screening by starting strain, and concrete screening step is as follows:
(1) preparation of bacteria suspension
The starting strain list bacterium colony grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 40 DEG C of cultivation 12h, after getting 1mL medium centrifugal, use brine twice, and resuspended with 9mL physiological saline;
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.Bacterium liquid through irradiating is coated lithium chloride flat board after gradient dilution, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 40 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle choose preserve to inclined-plane with colony diameter ratio the maximum, bacteria suspension is mixed with after purifying, fully mix with ethyl sulfate stoste after gradient dilution, and in 40 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board after gradient dilution;
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 40 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium Li-2013-A, Li-2013-02, Li-2013-B after purifying;
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-A obtained, Li-2013-02, Li-2013-B carries out shake flask fermentation in the 250mL shaking flask containing 30mL fermention medium, seed inoculum size 10% (V/V), 40 DEG C, 100r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid, measures crude enzyme liquid enzyme and lives, filtering out bacterial strain K1-1 is stable most superior strain, and enzyme is lived and reached 31500U/mL;
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%, lithium chloride 0.9%, agar 2%;
Described seed culture medium: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%;
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%;
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 fermentation culture 72h.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400728A (en) * | 2015-12-15 | 2016-03-16 | 重庆大学 | Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof |
| CN105567658A (en) * | 2016-03-10 | 2016-05-11 | 东北农业大学 | Method for carrying out mixed mutation on PLC (Phospholipase C) produced by bacillus cereus |
| CN105884547A (en) * | 2016-06-29 | 2016-08-24 | 兰赫(上海)生物科技有限公司 | Special leaf fertilizer for fruit trees and preparation method thereof |
| CN107325985A (en) * | 2017-07-13 | 2017-11-07 | 首都博物馆 | The production method and its application of bacillus subtilis and responsive to temperature type amylase |
| CN112226388A (en) * | 2020-10-23 | 2021-01-15 | 微米环创生物科技(北京)有限公司 | Novel enzyme-producing species of propionibacteriaceae and application thereof |
| CN116286746A (en) * | 2023-05-08 | 2023-06-23 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409006A (en) * | 2011-06-29 | 2012-04-11 | 林元山 | Strain and process method for producing acidic thermophilic amylase |
| CN102787130A (en) * | 2012-07-17 | 2012-11-21 | 天津科技大学 | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method |
| CN103224920A (en) * | 2013-02-04 | 2013-07-31 | 江南大学 | Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis |
-
2013
- 2013-11-14 CN CN201310566374.5A patent/CN104630084A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409006A (en) * | 2011-06-29 | 2012-04-11 | 林元山 | Strain and process method for producing acidic thermophilic amylase |
| CN102787130A (en) * | 2012-07-17 | 2012-11-21 | 天津科技大学 | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method |
| CN103224920A (en) * | 2013-02-04 | 2013-07-31 | 江南大学 | Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis |
Non-Patent Citations (1)
| Title |
|---|
| 王春铭等: "高温α-淀粉酶菌的产酶条件、酶性质与环境应用研究", 《环境科学学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400728A (en) * | 2015-12-15 | 2016-03-16 | 重庆大学 | Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof |
| CN105567658A (en) * | 2016-03-10 | 2016-05-11 | 东北农业大学 | Method for carrying out mixed mutation on PLC (Phospholipase C) produced by bacillus cereus |
| CN105884547A (en) * | 2016-06-29 | 2016-08-24 | 兰赫(上海)生物科技有限公司 | Special leaf fertilizer for fruit trees and preparation method thereof |
| CN107325985A (en) * | 2017-07-13 | 2017-11-07 | 首都博物馆 | The production method and its application of bacillus subtilis and responsive to temperature type amylase |
| CN107325985B (en) * | 2017-07-13 | 2019-12-27 | 首都博物馆 | Production method and application of bacillus subtilis and temperature-sensitive amylase |
| CN112226388A (en) * | 2020-10-23 | 2021-01-15 | 微米环创生物科技(北京)有限公司 | Novel enzyme-producing species of propionibacteriaceae and application thereof |
| CN116286746A (en) * | 2023-05-08 | 2023-06-23 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
| CN116286746B (en) * | 2023-05-08 | 2023-08-15 | 南京农丰生物科技有限公司 | Method for purifying amylase by liquid chromatography |
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