CN105400728A - Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof - Google Patents
Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof Download PDFInfo
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Abstract
The invention discloses a bacterial strain producing high-temperature-resistant beta-galactosidase and a screening method thereof and belongs to the technical field of biological chemistry and protein engineering. The screening method includes the specific and sequential steps of sample collection, primary screening, secondary screening of culture and beta-galactosidase activity assay. Mainly, soil of hot spring land is collected and boiled, then bacterial colonies are screened, and the technical problem how to produce high-enzyme-activity beta-galactosidase at high temperature is solved through a high-temperature culturing method. The bacterial strain is short in beta-galactosidase production period, high in beta-galactosidase production speed, good in genetic stability, thermal stability and pH stability, high in enzyme catalytic activity and high in production safety, and culturing conditions are simple. The bacterial strain has broad prospects in the fields of food industry and medical and biochemical analysis, in particular to the dairy industry.
Description
One, technical field
The present invention relates to a kind of bacterial strain and the screening method thereof that produce high temperature resistant beta-galactosidase enzymes, belong to technical field of biochemical industry, protein engineering.
Two, background technology
Beta-galactosidase enzymes (β-galactosidase), formal name used at school is β-D-galactoside galactohydrolase (β-D-galactoside, galactohydrolase, EC3.2.1.23), commodity are called Sumylact L (Lactase), can the hydrolysis reaction of β-Isosorbide-5-Nitrae-semi-lactosi glycosidic bond in catalysing lactose or lactose analog.
Beta-galactosidase enzymes is widely used in the fields such as foodstuffs industry, medical science and biochemical analysis, especially in dairy industry, plays great effect.Be usually used in being hydrolyzed the lactose in cow's milk and other milk-product, also can be used to generate oligomeric galactose, be widely used in the production of low-lactose milk and non-crystalline type enriching milk, by lactose degradation be glucose and semi-lactosi to produce low lactose milk, thus effectively solve the problems such as lactose intolerance.
Beta-galactosidase enzymes is mainly derived from animals and plants and microorganism.Because microorganism has the advantages such as the high and separation and purification of fast growth, metabolic efficiency is relatively simple, microorganism beta-galactosidase enzymes becomes the main source of industrialization zymin.But the Sumylact L zymologic property of different sources differs greatly, as the mould Sumylact L in the source such as aspergillus niger, aspergillus oryzae, catalytic temperature higher (40 ~ 55 DEG C), but optimal reaction pH value lower (pH3.0 ~ 4.5), be applicable to the process of cheese processing by product acid whey; The Sumylact L optimal reactive temperature (35 ~ 40 DEG C) near middle temperature of the yeast sources such as Kluyveromyces lactis and Kluyveromyces fragilis, and optimal reaction pH weakly acidic pH (pH6.5 ~ 7.0), be applicable to the production of low lactose milk (pH6.5 ~ 6.8) in milk processing: bacterium Sumylact L more complicated, optimal reactive temperature has from low temperature, midium temperature to high temperature and ultrahigh-temperature, but generally speaking optimal reaction pH all near neutral.
Current dairy industry obtains for many extraction after induction product enzyme from yeast, aspergillus tubigensis or milk-acid bacteria of beta-galactosidase enzymes reduced lactose.Although current many bibliographical informations screening and separating multiple beta-galactosidase strain, as the scheme of the serial bacterial strains such as yeast, genus lactubacillus, Bacillus, Eurotium, but produce the most optimum temperature of beta-galactosidase enzymes used at present at about 30 DEG C, enzyme activity is at high temperature all very low, and cost is high, be difficult to meet market and needed for producing, and all there is thermostability shortcoming good not enough, seriously limit its application in milk-product.If adopt high temperature resistant beta-galactosidase enzymes, can overcome the defect of poor heat stability, enzyme, at 60 DEG C and above temperature hydrolyzes lactose, can contribute to suppressing miscellaneous bacteria microbial growth.And enzyme reaction rate is fast, enzyme dosage is low, reaction times is short, can to carry out with pasteurize in production or cooling stages is incubated and is hydrolyzed simultaneously, namely in pasteur sterilizing process, carry out lactose hydrolysis simultaneously, lactose hydrolysis and pasteurization operation are united two into one, not only shortens the production cycle, and the waste heat that can make full use of pasteurization is to reduce energy consumption, thus significantly save production cost.Meanwhile, for its transportation problem, also its thermotolerance is claimed.
China according to epidemiological survey research show, mainly coloured race's ratio is higher for lactose intolerance, with Aisa people and Black American's incidence the highest.The people of 75% ~ 95% is about had to suffer from lactose intolerance in various degree in China resident.Therefore for Chinese, Sumylact L has the researching value and meaning that are even more important.Beta-galactosidase enzymes also has galactoside transferance, for the production of bifidus factor, thus widespread use in probiotic food is produced.Meanwhile, in January, 2015, two qi bifidus bacilluss (Biofidobacteriumbifidum) are listed in state food additive new variety.Beta-galactosidase enzymes is made to have larger effect by food-processing industry.
How to overcome the deficiencies in the prior art, accelerate the resistant to elevated temperatures beta-galactosidase enzymes product of research and development, become one of emphasis difficult problem to be solved of letter in current technical field of biochemical industry, there is very great meaning.Similar is CN103614313A as publication No., a kind of bacterial strain and screening method thereof of producing high temperature resistant beta-galactosidase enzymes of fermenting by name, and publication No. is CN102250856A, the structure of a kind of thermostable beta-galactosidase mutant by name, propose fermentation respectively and produce the bacterial strain of high temperature resistant beta-galactosidase enzymes and the construction process of screening method and thermostable beta-galactosidase mutant thereof, all how research is produced to resistant to elevated temperatures beta-galactosidase enzymes and has been made certain contribution, but bacterial strain whether high yield there is no clear and definite data with whether being easy to be separated.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art and a kind of bacterial strain and the screening method thereof that produce high temperature resistant beta-galactosidase enzymes are provided, this bacterial strain produces that cycle of beta-galactosidase enzymes is short, speed is fast, genetic stability good, Heat stability is good, pH good stability, enzyme catalysis are energetic, and culture condition is easy, production security is high, high temperature resistant.
According to the bacterial strain of a kind of high temperature resistant beta-galactosidase enzymes that the present invention proposes, it is characterized in that the Classification And Nomenclature of described bacterial strain Z89 is subtilis (Bacillussubtilis).
Above-mentioned purpose of the present invention is achieved by the following technical programs.According to a kind of screening method producing the bacterial strain of high temperature resistant beta-galactosidase enzymes that the present invention proposes, it is characterized in that in turn including the following steps:
Step one, sample collecting: pedotheque is adopted in hot spring and dairy farm soil;
Step 2, primary dcreening operation: 1 ~ 2g soil sample is placed in 10 ~ 20ml distilled water, does gradient dilution after mixing, pipettes 120 μ L bacterium liquid uniform application on screening culture medium flat board, is positioned in 30 DEG C of incubators and cultivates 24 ~ 48h, single bacterium colony line with blueness hydrolysis circle around picking from screening culture medium flat board, repeatedly rule 3 times, obtain pure growth by 1 ~ 3% in the line of 35 DEG C of solid mediums, 36 ~ 48h is cultivated at 35 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 35 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 45 DEG C of solid mediums, cultivate 36 ~ 48h, 3 ~ 5 the single bacterium colony liquid medium withins 45 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 55 DEG C of solid mediums, 36 ~ 48h is cultivated at 55 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 65 DEG C of solid mediums, 36 ~ 48h is cultivated at 65 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 75 DEG C of solid mediums, 36 ~ 48h is cultivated at 75 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 75 DEG C got wherein cultivate 12 ~ 24h, find that it does not grow, get 65 DEG C of bacterial strains still grown 1% ~ 3%, be inoculated in solid medium, cultivate 24 ~ 36h for 70 DEG C, find that it still can grow, so its optimum temperuture is at 65 DEG C ~ 70 DEG C.
Step 3, bacterial strain sieves again: by after primary dcreening operation through being separated, the bacterial strain that obtains of purifying respectively picking 2 ~ 3 articulating enter seed culture medium, activation 24 ~ 36h under the condition of 65 ~ 70 DEG C and 200 ~ 300r/min, being linked into the inoculum size of 2% (v/v) is equipped with in the 250ml triangular flask of 40ml fermention medium again, cultivates 24 ~ 36h with under identical condition; Frozen centrifugation (10000 ~ 12000r/min at 4 ~ 5 DEG C again, 10 ~ 15min) fermented liquid, after getting supernatant liquor and fragmentation, thalline measures the hydrolyzing activity of beta-galactosidase enzymes respectively, determine that beta-galactosidase enzymes is intracellular enzyme or extracellular enzyme, thus screening beta-galactosidase enzymes superior strain;
Step 4, the enzyme assay of beta-galactosidase enzymes: use colorimetric method for determining enzyme to live; 2 ~ 3mgONPG is dissolved in 2 ~ 3ml dipotassium hydrogen phosphate/potassium phosphate buffer, the enzyme liquid 0.5 ~ 2.5ml of suitably dilution is added after preheating, 15 ~ 20min is reacted at 55 ~ 65 DEG C, add the sodium carbonate solution 2.5 ~ 3.5ml of 0.15 ~ 0.25mol/L by reaction terminating, leave standstill 5 ~ 10min, survey the absorbance value of its product ONP at 420nm; The definition of beta-galactosidase enzymes enzymic activity: under these conditions, enzyme catalysis neck nitrophenol-β-D-galactoside (ONPG) is reacted 1min and is generated 1 μm of ol and lead enzyme amount needed for nitrophenol product (ONP).
The composition of described screening culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, X-gal0.035g/L, extractum carnis 10g/L, and agar 20g/L, condition is pH7.0.
The composition of described slant preservation substratum is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, agar 20g/L, and condition is pH7.0.
The composition of described seed culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, and condition is pH7.0.
The composition of described basal fermentation medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, calcium chloride 0.11g/L, manganous sulfate 0.001g/L, magnesium sulfate 0.3g/L, potassium primary phosphate 0.05g/L, ferrous sulfate 0.03g/L, and condition is pH7.0.
Outstanding advantages of the present invention and beneficial effect:
1. the present invention produce beta-galactosidase enzymes growth temperature be 65 ~ 70 DEG C, optimal reactive temperature is 65 DEG C, most high enzymatic activity high and survey that enzyme activity all lives at the highest enzyme about 90%; Enzyme is incubated 90min at 50 DEG C ~ 80 DEG C, and remnant enzyme activity, more than 85%, has excellent thermostability.
2. enzyme is between pH4.5 ~ 7.5, and enzyme is lived stable, and in view of the pH value of most of milk-product and beverage is also within the scope of this, therefore the beta-galactosidase enzymes that the biochemical characteristic of this enzyme makes it produce will have stronger application in milk-product processing.
3. beta-galactosidase enzymes of the present invention at high temperature has satisfactory stability, therefore in fields such as foodstuffs industry, medical science and biochemical analysises, especially in dairy industry, plays great effect.
Be below of the present inventionly a kind ofly produce the bacterial strain of high temperature resistant beta-galactosidase enzymes and the specific embodiment of screening method thereof, but the present invention is not limited.
Accompanying drawing explanation
Fig. 1 produces the dull and stereotyped schematic diagram of beta-galactosidase strain primary dcreening operation.
Fig. 2 high temperature resistant beta-galactosidase enzymes optimum temperature schematic diagram of the present invention
The thermostability schematic diagram of Fig. 3 high temperature resistant beta-galactosidase enzymes of the present invention.
The suitableeest action pH schematic diagram of Fig. 4 high temperature resistant beta-galactosidase enzymes of the present invention.
Fig. 5 high temperature resistant beta-galactosidase enzymes pH stability schematic diagram of the present invention.
Embodiment 1: the screening of producing high temperature resistant beta-galactosidase strain.
A kind of screening method producing the bacterial strain of high temperature resistant beta-galactosidase enzymes that the present invention proposes, is characterized in that comprising following concrete steps successively:
(1) sample collecting: some at hot spring and the dairy farm earth that fetches earth;
(2) primary dcreening operation: 1g soil sample is placed in 10ml distilled water, does gradient dilution after mixing, pipettes 120 μ L bacterium liquid uniform application on screening culture medium flat board, is positioned in 30 DEG C of incubators and cultivates 24 ~ 48h, single bacterium colony line with blueness hydrolysis circle around picking from screening culture medium flat board, repeatedly rule 3 times, obtain pure growth by 1 ~ 3% in the line of 35 DEG C of solid mediums, 48h is cultivated at 35 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 35 DEG C got wherein cultivate 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 45 DEG C of solid mediums, cultivate 48h, 3 ~ 5 the single bacterium colony liquid medium withins 45 DEG C got wherein cultivate 24h, getting 1% to be inoculated in solid medium in the line of 55 DEG C of solid mediums, 48h is cultivated at 55 DEG C, 3 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 65 DEG C of solid mediums, 48h is cultivated at 65 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 24h, to transfer after obtaining pure growth slant preservation, wherein, the composition of screening culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, X-gal0.035g/L, extractum carnis 10g/L, agar 20g/L, and condition is pH=7.0, sterilising temp 120 DEG C, sterilization time 20min, the composition of slant preservation substratum is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, agar 20g/L, and condition is pH=7.0, sterilising temp 120 DEG C, sterilization time 20min,
(3) bacterial strain sieves again: by after primary dcreening operation through being separated, the bacterial strain that obtains of purifying respectively picking 2 articulating enter seed culture medium, 36h is activated under 45 DEG C of conditions with 200r/min, being linked into the inoculum size of 2% (v/v) is equipped with in the 250ml triangular flask of 40ml fermention medium again, cultivates 24h with under identical condition; Frozen centrifugation (10000r/min at 4 DEG C again, 10min) fermented liquid, after getting supernatant liquor and fragmentation, thalline measures the hydrolyzing activity of beta-galactosidase enzymes respectively, determines that beta-galactosidase enzymes is intracellular enzyme or extracellular enzyme, thus screening beta-galactosidase enzymes superior strain; Wherein, the composition of seed culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, and condition is pH=7.0, sterilising temp 120 DEG C, sterilization time 20min;
The composition of basal fermentation medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, calcium chloride 0.11g/L, manganous sulfate 0.001g/L, magnesium sulfate 0.3g/L, potassium primary phosphate 0.05g/L, ferrous sulfate 0.03g/L, condition is pH=7.0, sterilising temp 120 DEG C, sterilization time 20min, add substratum after the independent sterilizing of lactose, the independent sterilising temp of lactose is 100 DEG C, sterilization time 15min;
(4) enzyme assay of beta-galactosidase enzymes: use colorimetric method for determining enzyme to live; 2mgONPG is dissolved in 2ml dipotassium hydrogen phosphate/potassium phosphate buffer, add the enzyme liquid 0.5ml of suitably dilution after preheating, at 55 DEG C, react 15min, add the sodium carbonate solution 2.5ml of 0.15mol/L by reaction terminating, leave standstill 5min, survey the absorbance value of its product ONP at 420nm; The definition of beta-galactosidase enzymes enzymic activity: under these conditions, enzyme catalysis neck nitrophenol-β-D-galactoside (ONPG) is reacted 1min and is generated 1 μm of ol and lead enzyme amount needed for nitrophenol product (ONP).
Embodiment 2: the Morphology and physiology biochemical character producing high temperature resistant beta-galactosidase strain.
The product beta galactose former times enzyme bacterial strain that screening method of the present invention obtains is after extractum carnis peptone culture medium flat plate cultivates 24h, and observe bacterium colony and thalli morphology, surface is more coarse, and edge is irregular, the dirty white of bacterium colony, and gramstaining is positive; The form of thalline is that quarter butt is bar-shaped; Thalline size is 0.65-0.7 μm of 2.5-3 μm again; At thalline, central authorities form endogenous spore, and after sporulation, thalline does not expand; Thalline without pod membrane, peritrichous, thalline has mobility; Gram-positive; Grow in liquid medium within, form wrinkle ether; Cellulose decomposition experiment is negative; In carbohydrate fermentation experiment, this bacterial strain can utilize Zulkovsky starch, glucose, fructose, lactose, wood sugar, maltose and sucrose to do uniqueness carbon source for growth; In other Physiology and biochemistries detect, this bacterial strain catalase is positive, V.P measures and is positive, energy hydrolyzed starch, casein, liquefy gelatin, can utilize Citrate trianion, nitrate reduction is positive, and litmus milk is also Antigen positive hybridomas, urea utilizes negative, does not produce fluorochrome, and methyl red experiment is positive; The morphology that bacterial strain Z89 is concrete and physiological and biochemical property are in table 1, bacillus subtilis in bacterium colony, dyeing characteristic and physiological and biochemical property and " uncle outstanding bacterium reflect handbook " (the 8th edition) is close, can determine that bacterial strain Z89 is subtilis (Bacillussubtilis).
Table 1 bacterial strain Z89 morphology and physio-biochemical characteristics
Embodiment 3: bacterial strain produce the enzymatic property of high temperature resistant beta-galactosidase enzymes.
1, the optimum temperature of beta-galactosidase enzymes:
Fermentation after enzyme liquid is centrifugal remove thalline after, get supernatant as crude enzyme liquid, for zymologic property preliminary study.The optimal reactive temperature of enzyme refers in differing temps, reaction pH7.0, with most high enzymatic activity for 100, calculates the enzyme activity under differing temps.Result draws, the optimal reactive temperature of this enzyme 65 DEG C, and meanwhile, this enzyme catalytic activity in higher temperature range is all higher, strong adaptability.
2, the thermostability of beta-galactosidase enzymes:
Beta-galactosidase enzymes crude enzyme liquid is placed in respectively differing temps (30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C), under the condition of pH7.0, insulation 180min, surveys enzyme every 30min sampling and lives, calculate respective residual enzyme activity, the enzyme activity of initial enzyme liquid is decided to be 100%, result is incubated 90min at 50 DEG C ~ 70 DEG C, and remnant enzyme activity, more than 80%, is incubated 60min at 60 DEG C and 70 DEG C, remnant enzyme activity is respectively 85% and 70%, show this bacterial strain produce beta-galactosidase enzymes there is strong thermostability.
3, the suitableeest action pH of beta-galactosidase enzymes:
Beta-galactosidase enzymes crude enzyme liquid carries out enzymatic reaction to measure its optimal pH under different pH (4.0 ~ 10.0), damping fluid used is: the sodium-acetate buffer of pH4.0 ~ 5.5, the sodium phosphate buffer of pH6.0 ~ 7.5, the Tris-HCl damping fluid of pH8.0 ~ 9.0, the Glycine-NaOH damping fluid of pH9.0 ~ 10.5, measuring temperature is 60 DEG C, with most high enzymatic activity for 100%, calculate the enzyme activity under different pH, the suitableeest action pH of result is 7, and between pH5.5 ~ 7.5, enzymic activity is more than 85%.
4, the ph stability of beta-galactosidase enzymes:
The mensuration of crude enzyme liquid pH stability is that enzyme liquid is hatched 1h in the damping fluid of different pH value at 40 DEG C, measures respective residual enzyme activity; Wherein will be decided to be 100 by most high enzymatic activity, result shows, keep the enzymic activity of more than 60%, and at more than pH7.5, enzymic activity then faster declines, and shows that beta-galactosidase enzymes is highly stable under slightly acidic and neutral environment between pH4.5 ~ 7.5.
These biochemical characteristics of this enzyme determine bacterial strain produce beta-galactosidase enzymes have larger application advantage in milk-product processing.
Claims (3)
1. produce a screening method for the bacterial strain of high temperature resistant beta-galactosidase enzymes, it is characterized in that, comprise the following steps:
(1) sample collecting: pedotheque is adopted in hot spring and dairy farm soil;
(2) primary dcreening operation: primary dcreening operation: 1 ~ 2g soil sample is placed in 10 ~ 20ml distilled water, does gradient dilution after mixing, pipettes 120 μ L bacterium liquid uniform application on screening culture medium flat board, is positioned in 30 DEG C of incubators and cultivates 24 ~ 48h, single bacterium colony line with blueness hydrolysis circle around picking from screening culture medium flat board, repeatedly rule 3 times, obtain pure growth by 1 ~ 3% in the line of 35 DEG C of solid mediums, 36 ~ 48h is cultivated at 35 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 35 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 45 DEG C of solid mediums, cultivate 36 ~ 48h, 3 ~ 5 the single bacterium colony liquid medium withins 45 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 55 DEG C of solid mediums, 36 ~ 48h is cultivated at 55 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 65 DEG C of solid mediums, 36 ~ 48h is cultivated at 65 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 65 DEG C got wherein cultivate 12 ~ 24h, getting 1 ~ 3% to be inoculated in solid medium in the line of 75 DEG C of solid mediums, 36 ~ 48h is cultivated at 75 DEG C, 3 ~ 5 the single bacterium colony liquid medium withins 75 DEG C got wherein cultivate 12 ~ 24h, find that it does not grow, get 65 DEG C of bacterial strains still grown 1% ~ 3%, be inoculated in solid medium, cultivate 24 ~ 36h for 70 DEG C, find that it still can grow, so its optimum temperuture is at 65 DEG C ~ 70 DEG C.To transfer after obtaining pure growth slant preservation;
(3) bacterial strain sieves again: by after primary dcreening operation through being separated, the bacterial strain that obtains of purifying respectively picking 2 ~ 3 articulating enter seed culture medium, activation 24 ~ 36h under the condition of 65 ~ 70 DEG C and 200 ~ 300r/min, being linked into the inoculum size of 2% (v/v) is equipped with in the 250ml triangular flask of 40ml fermention medium again, cultivates 24 ~ 36h with under identical condition; Frozen centrifugation (10000 ~ 12000r/min at 4 ~ 5 DEG C again, 10 ~ 15min) fermented liquid, after getting supernatant liquor and fragmentation, thalline measures the hydrolyzing activity of beta-galactosidase enzymes respectively, determine that beta-galactosidase enzymes is intracellular enzyme or extracellular enzyme, thus screening beta-galactosidase enzymes superior strain
(4) enzyme assay of beta-galactosidase enzymes: use colorimetric method for determining enzyme to live; 2 ~ 3mgONPG is dissolved in 2 ~ 3ml dipotassium hydrogen phosphate/potassium phosphate buffer, the enzyme liquid 0.5 ~ 2.5ml of suitably dilution is added after preheating, 15 ~ 20min is reacted at 55 ~ 65 DEG C, add the sodium carbonate solution 2.5 ~ 3.5ml of 0.15 ~ 0.25mol/L by reaction terminating, leave standstill 5 ~ 10min, survey the absorbance value of its product ONP at 420nm; The definition of beta-galactosidase enzymes enzymic activity: under these conditions, enzyme catalysis neck nitrophenol-β-D-galactoside (ONPG) is reacted 1min and is generated 1 μm of ol and lead enzyme amount needed for nitrophenol product (ONP).
The composition of described screening culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, X-gal0.035g/L, extractum carnis 10g/L, and agar 20g/L, condition is pH7.0.
The composition of described slant preservation substratum is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, agar 20g/L, and condition is pH7.0.
The composition of described seed culture medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, NaCl4g/L, and condition is pH7.0.
The composition of described basal fermentation medium is lactose 10g/L, Tryptones 5g/L, yeast extract 10g/L, calcium chloride 0.11g/L, manganous sulfate 0.001g/L, magnesium sulfate 0.3g/L, potassium primary phosphate 0.05g/L, ferrous sulfate 0.03g/L, and condition is pH7.0.
2. a kind of screening method producing the bacterial strain of high temperature resistant beta-galactosidase enzymes according to claims 1, is characterized in that, the sample of collection comes from hot spring ground and diary farm soil, and carries out boiling process before primary dcreening operation.
3. a kind of bacterial strain producing high temperature resistant beta-galactosidase enzymes according to claim 1, is characterized in that described bacterial strain has following characteristic:
(1) growth conditions: produce the growth temperature 65 ~ 70 DEG C of beta-galactosidase enzymes, optimum growth temperature 65 DEG C;
(2) enzymic activity: be incubated 90min at 50 ~ 80 DEG C, remnant enzyme activity, more than 85%, is incubated 60min at 80 DEG C and 90 DEG C, and remnant enzyme activity is respectively 85% and 70%;
(3) slightly acidic: enzymic activity is more than 85% between pH5.5 ~ 7.5, between pH4.5 ~ 7.5, keep the enzymic activity of more than 80%, the suitableeest action pH is 7.
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Cited By (4)
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|---|---|---|---|---|
| CN108977386A (en) * | 2018-08-08 | 2018-12-11 | 大连大学 | A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain |
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