CN110257305A - The breeding method of the Shewanella of high yield alkali protein - Google Patents
The breeding method of the Shewanella of high yield alkali protein Download PDFInfo
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Abstract
The present invention provides the breeding method of the Shewanella of high yield alkali protein, belongs to field of biotechnology, comprising: obtains the Shewanella bacterial strain of amino acid assembling modification；And the in-depth for making bacterial strain carry out intermittent low carbon, low nitrogen fermentation is cultivated；Above-mentioned intermittent low carbon, low nitrogen fermentation adds replenishers into the cultivation system for cultivating the predetermined amount time by in-depth cultivation period, at least one predetermined time, at the same secondary cryogenic strong alkali environment and realize.Breeding method provided by the invention can reduce the phenomenon that modified amino acid crosslinks compactness extent prevents infiltration to avoid the reduction of phage surface adsorption capacity, reduce containment and inhibition of the environment to albumen enzymatic synthesis, the inhibition that reduction metabolite breeds thallus is to increase cell density, improve the output and production efficiency of alkali protease, enhance the ability of the high salinity of albumen enzyme adaptation low temperature highly basic, it promotes enzyme low temperature active and thermal stability, produced alkali protease belongs to the low temperature resistant albuminoid of resistance to highly basic enzyme.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the breeding method of the Shewanella of high yield alkali protein.
Background technique
Alkali protease (Alkaline protease) refer under alkaline condition can aminosal peptide bond enzyme,
Its optimal pH is 9-11, and activated centre ser belongs to serine protease, not only can hydrolysising peptide key, also have hydrolysis of ester bonds with,
Amido bond and transesterification and the function of turning peptide, are widely present in animal and plant and microbial body, are a kind of very important industrial
Enzyme.Alkali protease application is very extensive, extensive in fields such as enzyme detergent, silk, feed, process hides, food, medicine, environmental protection
Using, 65% or more of industrial enzyme preparation market is accounted in worldwide, have critically important industry and economic value.
Microorganism is allowed into due to the features such as speed of growth is fast, growth conditions is special compared with simple, metabolic process and distribution is wide
For the important sources of protease.Microbial protease is ectoenzyme, is extracted from microorganism, not by resource, environment and sky
Between limitation, with animal protease and the incomparable superiority of plant rennet.The Producing Strain of alkali protease at present
Strain still concentrates on hay bacillus category, and source is more single, causes current protease structure also more single.Alkali protease
In addition to not being able to satisfy wanting for industrial development the problems such as there is single variety, also not high, expensive just like the activity of enzyme
It asks, large-scale industry mainly also relies on import with alkali protease, therefore the screening and breeding of high-quality bacterium producing multi enzyme preparation have ten
Divide important application value.
The protease of marine source is increasingly becoming research hotspot at present, since marine microorganism and terrestrial microbial are than it
Living environment is more severe, such as hypertonic, low temperature, high pressure, low nutrition, also brings up its unique metabolic pathway, therefore ocean is micro-
Biology may produce the protease with more unique zymologic property and structure, as ocean enzyme have wide action pH range,
Optimal pH and operative temperature is moderate, enzymatic activity with the reduction of reaction temperature declines the features such as slow, thus produce alkali protease
Marine microorganism have research and application value.
Shewanella belongs to facultative anaerobic bacteria, and in widely distributed Yu Haiyang and fresh water environment, most of bacterial strain belongs to γ-change
Shape bacterium door is in Gram-negative, and general length is 2-3 μm, and 0.4-0.7 μm of diameter of corynebacteria is moved by unipolarity flagellum
Driving.In a natural environment, Shewanella can with other type microbial fermentations be metabolized organic matter product, as formic acid, lactic acid,
Amino acid etc. maintains own metabolism as carbon source.It can also be to produce the hydrogen generated in hydrogen microbial metabolism in environment
Electron donor.Under anaerobic, Shewanella there is the oxidation of coupling organic substrates to restore with extracellular insoluble petal different
Change the ability of metal reduction, this feature makes it have stronger survival ability in different ecological environment, to its metabolite
As the zymologic property of alkali protease has an impact with structure, thus have to the research of alkali protease and Shewanella potential
Application value.
Summary of the invention
That the purpose of the present invention is to provide a kind of yield of enzyme is big, enzymatic activity is high, and it is fine and close to reduce modified amino acid crosslinks
The phenomenon that degree prevents infiltration to avoid the reduction of phage surface adsorption capacity, reduces containment and suppression of the environment to albumen enzymatic synthesis
Production is used, and reduces the inhibition that metabolite breeds thalli growth to increase cell density, is improved bacterial strain and is metabolized alkali protease
Output and production efficiency, can using stress effect enhancing the high salinity of albumen enzyme adaptation low temperature highly basic ability, promoted enzyme it is low
The breeding method of the Shewanella of the high yield alkali protein of warm activity and thermal stability, produced alkali protease belongs to low temperature resistant
The albuminoid of resistance to highly basic enzyme.
The technical solution that the present invention is taken to achieve the above object are as follows:
The breeding method of the Shewanella of high yield alkali protein, including, obtain the Shewanella of amino acid assembling modification
Bacterial strain；With, make bacterial strain carry out intermittent low carbon, low nitrogen fermentation in-depth cultivate；Above-mentioned intermittent low carbon, low nitrogen fermentation is by depth
Change in cultivation period, adds replenishers into the cultivation system for cultivating the predetermined amount time at least one predetermined time, while attached
Add low temperature strong alkali environment and realizes.This method first carries out surface-assembled modification to bacterium with amino acid, changes the complete of its cell membrane
Whole property or permeability improve the output and enzyme of protease then by the addition of intermittent low carbon, low nitrogen fermentation and replenishers
Vigor, and using the stress effect of thalli growth the adaptability of the high salinity of low temperature highly basic of protease is enhanced, Xi Washi
Bacterium yield of enzyme after cultivating is big, enzyme activity is high, and pH, temperature and the salinity scope of application of gained protease are extended, and has good
Good low temperature active and thermal stability, can reduce production cost, obtain good economic benefit.
In embodiments of the invention, the preparation step of the Shewanella bacterial strain of basic amino acid assembling modification are as follows: will
Amino acid is dispersed in the electrolyte solution that pH is 8-9, then adds Shewanella bacterial strain with the ratio of 10-20g/L, then
Crosslinking agent is added thereto, and shaking table concussion reaction 1-2h is then carried out to obtain the final product with the revolving speed of 150-300r/min.By band in system
The amino acid of positive electricity and amido functional group is assembled with electronegative Shewanella, can improve Shewanella surface amino groups function
Group quantity, both can increase the adsorption capacity on bacterial strain surface, while also changing the permeability of strain cell film surface, be conducive to by
In culture medium substance absorption and easily to bacterial strain internal transmission, and can from external source environment offer albumen enzymatic synthesis basic amine group
Acid synthesizes protease biological with this and carries out analog induction, changes metabolic pathway relevant with protease in promotion bacterial strain, makes
Bacterial strain is carried out to the direction for producing protease, so as to improve the output and enzyme activity of protease.
In more particular embodiment of the present invention, amino acid is basic amino acids lysine or arginine；Amino acid exists
Concentration in mixed system is 2-5wt%.It is furthermore preferred that amino acid is arginine.Arginine and lysine isoelectric point are larger, belong to
In basic amino acid, it is assembled in and is more advantageous to the transformation for promoting the relevant proenzyme of bacterial strain neutral and alkali protease to enzyme on bacterial strain, make
Bacterial strain is metabolized to the direction of alkali protease.
In more particular embodiment of the present invention, crosslinking agent additive amount is the 0.5-1.5% of bacterial strain weight；Crosslinking agent packet
1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine are included, wherein the weight ratio of 1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine is 2:
0.3-0.8:0.1-0.5.Collaboration can make to be cross-linked with each other between the amino acid for completing assembling with bacterium surface between crosslinking agent, then exist
Bacterium surface forms mucous membrane to carry out capsulation, and the thallus after capsulation is conducive to thallus convenient for collecting, keeping and reuse
Stability is kept in extraneous environmental catastrophe, mucous membrane has slow releasing function to metal ion, reduces it to bacterium or secretion gained egg
The inhibition of white enzyme and damaging action, wherein 5- chloro pentane acid and phosphatic hydroxylamine can utilize different space structures in amino acid crosslinks
Type reduces the compactness extent between amino acid structure, avoids amino acid crosslinks structure because of the difference of osmotic pressure inside and outside in the medium
And the phenomenon that causing adsorption capacity to reduce and then preventing infiltration in culture basal orientation bacterium, while shape between the two energy and intracellular protein
At hydrogen bond, disulfide bond is replaced both to can increase the enzyme of protease to maintain the structural stability of the enzyme of bacterial secretory using hydrogen bond
It is living, and can increase the stability of enzyme at low temperature.
In embodiments of the invention, in-depth cultivate operation are as follows: cultivation period 7-10d, wherein every 20-24h to
Replenishers are added in cultivation system, at the same by nurturing an environment by pH be 6.5-8, temperature is 25-40 DEG C and is adjusted to low temperature highly basic ring
Border, continues after cultivating 3-6h, nurturing an environment is adjusted to normal nurturing an environment and continues to cultivate, with training when adding replenishers every time
The adjusting of environment is educated, until cultivation period terminates.The various metabolites such as including protease can be secreted during strain growth, it is original
Culture medium belongs to low carbon, low nitrogen type, can preferentially utilize the basic amino acid of surface modification, while avoiding end products and rich nitrogen
Environment contains that low-carbon can be to avoid glucose metabolism product to the inhibiting effect of albumen enzymatic synthesis to the feedback of albumen enzymatic synthesis.
It under the damage effect of of short duration low temperature strong alkali environment, can excite bacterial strain that stress protect the comprehensive function with stimulating effect, be promoted
Thallus low temperature strong alkali environment survival rate, and utilize stress effect, enhance the produced protease of thallus under low temperature strong alkali environment
Adaptability.
In embodiments of the invention, low temperature strong alkali environment operating condition are as follows: temperature is 15-25 DEG C, pH 9-12.?
Bacterial strain deepens the nurturing period using low temperature strong alkali environment, and bacterium can be made extraneous bad to fight in growth period generation stress reaction
Environment can especially slow down the content of soluble protein that stress be injured and significantly rise, so that bacterium divides protease
The amount of secreting increases, and protease adapts to the environment of low temperature highly basic, is conducive to expand protease use scope, increases it using valence
Value.
In embodiments of the invention, the single use amount of replenishers is the 0.4-1% of culture solution weight；In replenishers
Contain sucrose, casein and ammonium chloride, weight ratio 1:0.5-1:0.2-0.5.Low carbon, low nitrogen type culture medium is unfavorable for thallus
Flourish, therefore intermittent iron supplement sugar source and nitrogen source, enable bacterial strain Rapid development and breeding, increase cell density, to numerous
It grows after consuming part carbon source and nitrogen source, containment effect reduces, and thallus accelerates synthesis secretion metabolite, can effectively increase alkalinity
The output of protease improves the production efficiency of protease.
In embodiments of the invention, breeding method further includes stabilizing to cultivate；Stabilize the step of cultivating are as follows: will be deep
Change the microorganism collection after cultivating, and access in 2216E sea water medium, Yu Wendu 20-45 DEG C, pH stablize under conditions of being 8-12
Change after cultivating 2-3d, is centrifuged and collects respectively and cultivate gained thallus and liquid.Stabilisation cultivation is carried out to thallus, portion can be made
Divide thallus to complete self-dissolving, release endocellular enzyme, while filtering out the thallus for adapting to alkaline environment, secondary cultivation or guarantor can be carried out
It deposits, is conducive to the scope of application for expanding bacterium, and the ability that bacterial strain produces alkali protease can be improved, can increase using sea water medium
The tolerance of salinity of bacterial strain, gained Shewanella bacterial strain enzymatic activity is high, yield of enzyme is big, and has good pH stability and thermostabilization
Property.
The present invention also provides the above methods to cultivate purposes of the resulting Shewanella in alkali protease production.This hair
The breeding method of the Shewanella production alkali protease of bright offer, produced prolease activity is high, and pH, temperature and the salinity of enzyme are suitable
It is extended with range, production cost can be reduced, obtain good economic benefit.
In embodiments of the invention, the applicable pH of alkali protease is 8-14, and Applicable temperature is 15-50 DEG C, is belonged to
The low temperature resistant albuminoid of resistance to highly basic enzyme.The optimum temperature of the alkali protease is 40 DEG C, and most suitable action pH is that 13,500mL shakes
Bottle fermentation enzyme activity is up to 2370-2800U/ml；Keep the temperature 1h under the conditions of pH8-14,40 DEG C, remnant enzyme activity 88% or more,
15-50 DEG C, keep the temperature 1h under the conditions of pH13, remnant enzyme activity increases the use scope of enzyme 75% or more.
The invention has the benefit that
1) surface-assembled modification first is carried out to bacterium with amino acid in the present invention, changes the integrality or infiltration of its cell membrane
Property, then by the addition of intermittent low carbon, low nitrogen fermentation and replenishers, the output and enzyme activity of protease are improved, and can subtract
The suppression of little end product and rich nitrogen environment to the feedback containment of albumen enzymatic synthesis and glucose metabolism product to albumen enzymatic synthesis
Production is used；
2) present invention can improve Shewanella surface amino groups number of functional groups using basic amino acid assembling modified bacteria, both
Can increase bacterial strain adsorption capacity, reduce the compactness extent between amino acid structure to avoid the reduction of phage surface adsorption capacity and
The phenomenon that preventing infiltration in culture basal orientation bacterium, and disulfide bond can be replaced using hydrogen bond, change and promote protease phase in bacterial strain
The metabolic pathway of pass, improves the output and enzyme activity of bacterial strain metabolism alkali protease, while increasing the stabilization of enzyme at low temperature
Property；
3) type of rearing of the intermittent addition replenishers and secondary cryogenic strong alkali environment of the present invention, both can increase phage surface
Permeability, facilitate thallus to obtain energy, reduce bad containment effect, and the suppression that metabolite breeds thalli growth can be reduced
System improves the yield and yield of protease, and by the stress effect of external environment, so that bacterium to increase thallus population densities
The middle content of soluble protein that stress be injured that can slow down significantly rises, and causes the secretory volume of protease to increase, improves albumen
The production efficiency of enzyme, and protease low temperature highly basic adaptive faculty is enhanced；
4) the high salinity adaptability enhancing of the low temperature highly basic of alkali protease obtained by breeding method provided by the invention, albumen
The low temperature active and thermal stability of enzyme get a promotion, and thallus yield of enzyme is big, enzymatic activity is high, and pH, temperature and the salinity of enzyme are applicable in
Range is extended, and production cost can be reduced.
Present invention employs above-mentioned technical proposals to provide the breeding method of the Shewanella of high yield alkali protein, compensates for
The deficiencies in the prior art, reasonable design, easy operation.
Detailed description of the invention
Fig. 1 is influence schematic diagram of the different breeding methods to Shewanella cell density；
Fig. 2 is alkali protease optimal reactive temperature test result schematic diagram；
Fig. 3 is that the thermal stability of alkali protease changes schematic diagram；
Fig. 4 is the enzyme activity change curve of alkali protease at low ambient temperatures；
Fig. 5 is alkali protease optimal pH test result schematic diagram；
Fig. 6 is the pH stability change schematic diagram of alkali protease.
Specific embodiment
Technical solution of the present invention is described in further detail below in conjunction with specific embodiment and attached drawing:
In specific embodiment of the present invention, it is soluble starch culture medium that low carbon, low nitrogen culture medium used is cultivated in in-depth,
Including soluble starch 25g, yeast extract 5g, beef extract 10g, peptone 15g, glucose 1g, sodium chloride 5g, two hydration chlorine
Change calcium 0.2g, potassium hydrogen phosphate 3.5g, sodium dihydrogen phosphate 0.3g, sodium carbonate 0.56g, Chen Haishui 1000mL.
In specific embodiment of the present invention, stabilizing and cultivating used medium is 2216E sea water medium, the culture medium
In include peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, agar 20g, Chen Haishui 1000mL.
Embodiment 1:
The breeding method of the Shewanella of high yield alkali protein, including, obtain the Shewanella of amino acid assembling modification
Bacterial strain；With, make bacterial strain carry out intermittent low carbon, low nitrogen fermentation in-depth cultivate；Above-mentioned intermittent low carbon, low nitrogen fermentation is by depth
Change in cultivation period, adds replenishers into the cultivation system for cultivating the predetermined amount time at least one predetermined time, while attached
Add low temperature strong alkali environment and realizes.This method first carries out surface-assembled modification to bacterium with amino acid, changes the complete of its cell membrane
Whole property or permeability improve the output and enzyme of protease then by the addition of intermittent low carbon, low nitrogen fermentation and replenishers
Vigor, and using the stress effect of thalli growth the adaptability of the high salinity of low temperature highly basic of protease is enhanced, Xi Washi
Bacterium yield of enzyme after cultivating is big, enzyme activity is high, and pH, temperature and the salinity scope of application of gained protease are extended, and has good
Good low temperature active and thermal stability, can reduce production cost, obtain good economic benefit.
The preparation step of the Shewanella bacterial strain of above-mentioned basic amino acid assembling modification are as follows: amino acid is dispersed in pH
For in 8 electrolyte solution, Shewanella bacterial strain is then added with the ratio of 15g/L, then add crosslinking agent thereto, then with
The revolving speed of 250r/min carries out shaking table concussion reaction 1h to obtain the final product.By positively charged in system and amido functional group amino acid and with negative
The Shewanella of electricity is assembled, and can be improved Shewanella surface amino groups number of functional groups, both can increase the absorption on bacterial strain surface
Capacity, while the permeability of strain cell film surface is also changed, be conducive to by substance absorption in culture medium and easily to bacterium
Strain internal transmission, and the basic amino acid of albumen enzymatic synthesis can be provided from external source environment, protease biological is synthesized with this and is carried out
Analog induction changes metabolic pathway relevant with protease in promotion bacterial strain, carries out bacterial strain to the direction for producing protease, with
This improves the output and enzyme activity of protease.
Above-mentioned amino acid is basic amino acids lysine or arginine；Concentration of the amino acid in mixed system is
2.5wt%.Amino acid is arginine in the present embodiment.Arginine and lysine isoelectric point are larger, belong to basic amino acid, assembling
It is more advantageous to the transformation for promoting the relevant proenzyme of bacterial strain neutral and alkali protease to enzyme on bacterial strain, makes bacterial strain to alkali protease
Direction metabolism.
Above-mentioned crosslinking agent additive amount is the 1.1% of bacterial strain weight；Crosslinking agent includes 1,2- ethylenediamine, 5- chloro pentane acid and phosphoric acid
Azanol, wherein the weight ratio of 1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine is 2:0.5:0.4.Between crosslinking agent collaboration can make with
Bacterium surface is completed to be cross-linked with each other between the amino acid assembled, then forms mucous membrane in bacterium surface to carry out capsulation, packing
Thallus after change is conducive to thallus and keeps stability, mucous membrane pair in extraneous environmental catastrophe convenient for collecting, keeping and reuse
Metal ion has slow releasing function, reduces its inhibition and damaging action to bacterium or secretion gained protease, wherein 5- chloro pentane acid
With phosphatic hydroxylamine the compactness extent between amino acid structure can be reduced, is avoided in amino acid crosslinks using different steric configurations
Amino acid crosslinks structure inside and outside in the medium the difference of osmotic pressure due to cause adsorption capacity to reduce and then prevent culture basal orientation
In bacterium the phenomenon that infiltration, while hydrogen bond is formed between the two energy and intracellular protein, replaces disulfide bond using hydrogen bond, to maintain
The structural stability of the enzyme of bacterial secretory, not only can increase the enzyme activity of protease, but also can increase the stability of enzyme at low temperature.
Operation is cultivated in above-mentioned in-depth are as follows: cultivation period 7d, wherein adding replenishers into cultivation system every 20h, together
When by nurturing an environment by pH be 7, temperature is 30 DEG C and is adjusted to low temperature strong alkali environment, continue after cultivating 4h, nurturing an environment adjusted
Continue to cultivate for normal nurturing an environment, with the adjusting of nurturing an environment when adding replenishers every time, until cultivation period terminates.Bacterium
Strain growth period can secrete the various metabolites such as including protease, and original culture medium belongs to low carbon, low nitrogen type, can preferential benefit
With the basic amino acid of surface modification, while end products and rich nitrogen environment being avoided to contain the feedback of albumen enzymatic synthesis, low-carbon
It can be to avoid glucose metabolism product to the inhibiting effect of albumen enzymatic synthesis.In the damage effect of of short duration low temperature strong alkali environment
Under, it can excite bacterial strain that stress protect the comprehensive function with stimulating effect, promote thallus in the survival rate of low temperature strong alkali environment, and benefit
With stress effect, enhance adaptability of the produced protease of thallus under low temperature strong alkali environment.
Above-mentioned low temperature strong alkali environment operating condition are as follows: temperature is 22 DEG C, pH 10.5.It is used in the bacterial strain in-depth nurturing period
Low temperature strong alkali environment can make bacterium generate stress reaction in growth period to fight extraneous poor environment, can especially slow down and answer
The content of soluble protein for swashing injury significantly rises, so that bacterium increases the secretory volume of protease, and protease energy
The environment for adapting to low temperature highly basic is conducive to expand protease use scope, increases its application value.
The single use amount of above-mentioned replenishers is the 0.8% of culture solution weight；Contain sucrose, casein and chlorine in replenishers
Change ammonium, weight ratio 1:0.6:0.3.Low carbon, low nitrogen type culture medium is unfavorable for thallus flourish, therefore intermittent iron supplement sugar
Source and nitrogen source, enable bacterial strain Rapid development and breeding, increase cell density, after breeding and consuming part carbon source and nitrogen source,
Containment effect reduces, and thallus accelerates synthesis secretion metabolite, can effectively increase the output of alkali protease, improve protease
Production efficiency.
Above-mentioned breeding method further includes stabilizing to cultivate；Stabilize the step of cultivating are as follows: the thallus after cultivating in-depth is received
Collect, and access in 2216E sea water medium, 35 DEG C of Yu Wendu, pH stabilize cultivation 2d under conditions of being 10.5 after, is centrifuged and simultaneously divides
Gained thallus and liquid Shou Ji not cultivated.Stabilisation cultivation is carried out to thallus, part thallus can be made to complete self-dissolving, released
Endocellular enzyme, while the thallus for adapting to alkaline environment is filtered out, secondary cultivation or preservation can be carried out, is conducive to expand being applicable in for bacterium
Range, and the ability that bacterial strain produces alkali protease can be improved, it can increase the tolerance of salinity of bacterial strain, gained Shiva using sea water medium
Salmonella bacterial strain enzymatic activity is high, yield of enzyme is big, and has good pH stability and thermal stability.
The above method cultivates purposes of the resulting Shewanella in alkali protease production.Xi Washi provided by the invention
Bacterium produces the breeding method of alkali protease, and produced prolease activity is high, and pH, temperature and the salinity scope of application of enzyme are expanded
Exhibition, can reduce production cost, obtain good economic benefit.The applicable pH of above-mentioned alkali protease is 8-14, and Applicable temperature is
15-50 DEG C, belong to the low temperature resistant albuminoid of resistance to highly basic enzyme.The optimum temperature of the alkali protease is 40 DEG C, most suitable action pH
2370-2800U/ml is up to for 13,500mL shake-flask fermentation enzyme activity；1h is kept the temperature under the conditions of pH8-14,40 DEG C, remnant enzyme activity exists
88% or more, 1h is kept the temperature under the conditions of 15-50 DEG C, pH13, remnant enzyme activity increases the use scope of enzyme 75% or more.
Embodiment 2:
The breeding method of the Shewanella of high yield alkali protein, the specific steps are as follows:
(1) arginine is dispersed in the electrolyte solution that pH is 8.5, is configured to the mixing that concentration is 3.5wt%
Then system is in the Shewanella bacterial strain of logarithmic growth phase with the ratio addition of 15g/L, then adds and account for bacterial strain weight 0.8%
Crosslinking agent, shaking table concussion reaction 1.5h is then carried out to get the Xi Washi of amino acid assembling modification with the revolving speed of 200r/min
Bacteria strain, the weight ratio of 1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine that above-mentioned crosslinking agent includes are 2:0.3:0.2；
(2) bacterial strain by amino acid assembling modification accesses in soluble starch culture medium, and adjusts bacterial strain access amount and be
OD600=0.5, then start the in-depth that the period is 10d under conditions of pH is 7.5, temperature is 35 DEG C and cultivate, every for 24 hours to training
Educate in system the replenishers that addition accounts for culture solution weight 0.5%, at the same by nurturing an environment be adjusted to temperature be 20 DEG C, pH 11,
Continue after cultivating 4.5h, nurturing an environment is adjusted to pH is 7.5, temperature is 35 DEG C, with cultivating ring when adding replenishers every time
The adjusting in border, until cultivation period terminates, by medium centrifugal, collecting liquid is crude enzyme liquid, and sucrose is contained in replenishers, is done
Casein and ammonium chloride, weight ratio 1:0.7:0.2；
(3) thallus being collected by centrifugation is accessed in 2216E sea water medium, under conditions of 40 DEG C of Yu Wendu, pH are 12
It stabilizes and cultivates 3d, the culture that thallus saves or enters subsequent cycle is collected in centrifugation, and centrifugation gained liquid is complex liquid；
(4) crude enzyme liquid and complex liquid are mixed to form multiple enzyme solution, then multiple enzyme solution is concentrated by ultrafiltration apparatus, then
By concentrate vacuum freezedrying to get alkali protease, the cultivation period for being considered as Shewanella production alkali protease terminates.
Embodiment 3:
The present embodiment is optimized on the basis of embodiment 2, and specific Optimized Measures are as follows: step (2) supplement used
It further include the diethylene glycol of 0.1wt% and the dimethyl sulfoxide of 0.05wt% in agent, with the increase for cultivating the time, thalli growth
A large amount of stickums can be secreted in the process and are gathered in phage surface, be unfavorable for thalli growth and carry out energy exchange with culture solution,
Diethylene glycol and dimethyl sulfoxide in replenishers can pass rapidly through stickum, and be dispersed stickum in using hydrophily
In culture solution, the inhibition that metabolite breeds thalli growth is reduced, the permeability of phage surface is increased, thallus is facilitated to obtain energy
Amount improves the yield and yield of protease, and on the other hand the two can cooperate with the amino acid of bacterium surface to protease protein texture
As having an impact so that protein structure flexibility reduce and rigidity reinforced improves so that the acclimatization to cold ability of protease enhances
The low temperature active and thermal stability of protease；
It is consistent in other steps and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Embodiment 4:
The present embodiment and embodiment 2 the difference is that: the Shewanella bacterial strain of logarithmic growth phase is directly accessed depth
Change cultivation to be cultivated in low carbon, low nitrogen culture medium, without doing amino acid assembling modification；
It is consistent in other steps and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Embodiment 5:
The present embodiment and embodiment 2 the difference is that: crosslinking agent used is 1,2- ethylenediamine in step (1), is not added
Add 5- chloro pentane acid and phosphatic hydroxylamine；
It is consistent in other steps and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Embodiment 6:
The present embodiment and embodiment 2 the difference is that: in step (2) after bacterial strain addition replenishers, non-secondary cryogenic
Strong alkali environment；It is consistent in other steps and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Embodiment 7:
The present embodiment and embodiment 2 the difference is that: specific step is as follows for step (2): amino acid being assembled and is modified
Bacterial strain access soluble starch culture medium in, and adjust bacterial strain access amount be OD600=0.5, then pH be 8.5, temperature
Start the in-depth that the period is 10d under conditions of being 35 DEG C to cultivate, then by medium centrifugal, collecting liquid is crude enzyme liquid；Its
It is consistent in his step and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Embodiment 8:
The present embodiment and embodiment 2 the difference is that: specific step is as follows for step (2): amino acid being assembled and is modified
Bacterial strain access soluble starch culture medium in, and adjust bacterial strain access amount be OD600=0.5, then pH be 8.5, temperature
Start the in-depth that the period is 10d under conditions of being 35 DEG C to cultivate, then by medium centrifugal, collecting liquid is crude enzyme liquid, on
The replenishers for being 4.5% containing weight accounting in culture medium are stated, contain sucrose, casein and ammonium chloride, weight in replenishers
Than for 1:0.7:0.2；It is consistent in other steps and embodiment 2, complete the cultivation of the Shewanella of high yield alkali protein.
Test example 1:
Basic protein enzyme activity determination and pilot production
(1) basic protein enzyme activity determination: test specimen is that embodiment 1,2,3,4,5,7 and 8 cultivates gained Shewanella
Multiple enzyme solution, every group 5 are parallel, as a control group with embodiment 8.Measuring method: Folin reagent color developing method: 1mL enzyme solution is 40
DEG C, pH11, per minute hydrolysis generate 1 μ g tyrosine required for enzyme amount be 1 enzyme activity unit (U/mL).Steps are as follows: will
After sample answers enzyme solution progress 5000r/min centrifugation removal thallus, supernatant is taken to be diluted to borax-sodium hydroxide buffer solution
1mL enzyme solution is added as enzyme solution to be measured in debita spissitudo in test tube, casein solution 1mL is added, after shaking up in 40 DEG C of warm bath 2min
40 DEG C of warm bath 10min are added 2mL solution of trichloroacetic acid, shake up that (trichloroacetic acid is first added in blank group, and it is molten to add casein
Liquid).Static 10min is taken out, qualitative filter paper filters at a slow speed.1mL filtrate is taken, sodium carbonate liquor 5mL is added, adds Folin reagent to use molten
Liquid 1mL, 40 DEG C of colour developing 20min measure absorbance with 10mm cuvette in 680nm wavelength.Calculation method: U=A × K × 4/
(10 × n), wherein U indicates enzyme activity, and A indicates that light absorption value, K are the coefficient of standard curve, and 4 indicate reaction total volume, and 10 indicate anti-
Between seasonable, n is extension rate.The yield of alkali protease obtained by each embodiment is separately counted, as a result and statistical analysis is such as the following table 1 institute
Show.
1 basic protein enzyme activity determination of table and output statistics result
| Average enzyme activity U/mL | Percentage % is improved compared with control group | Yield mg | |
| Embodiment 1 | 2715.7 | 22.5 | 329.3 |
| Embodiment 2 | 2729.3 | 23.2 | 321.7 |
| Embodiment 4 | 2415.2 | 9.0 | 278.5 |
| Embodiment 5 | 2517.6 | 13.6 | 292.4 |
| Embodiment 7 | 2283.7 | 3.1 | 227.9 |
| Embodiment 8 | 2215.6 | —— | 246.5 |
As seen from the above table, enzyme activity difference is little between Examples 1 and 2, and enzyme activity improves 20% or more, embodiment 4
In contrast with 5, enzyme activity increasing value is poor, illustrates although the breeding method of embodiment 4 and 5 simplifies step, but cultivate
Much not as good as Examples 1 and 2, the enzyme activity of embodiment 7 and embodiment 8 is not much different the produced protease enzyme activity of bacterial strain, implements
The breeding method of example 1 and 2 more can protease produced to bacterial strain enzyme activity generate beneficial effect；Examples 1 and 2 production of enzyme difference
It is smaller, and it is significantly higher than other groups, it is that induction is not played to basic protein enzymatic synthesis due to amino acid unassembled in embodiment 4
It acts on, the difference of crosslinking agent makes amino acid crosslinks structure affect the growth and breeding of thallus in embodiment 5, and then affects enzyme
Yield, although 7 low carbon, low nitrogen environmental benefits of embodiment in albumen enzymatic synthesis, are unfavorable for thalli growth breeding, so yield compared with
Low, embodiment 8 is then bred due to being conducive to thallus under carbon nitrogen abundance environment, but plays the role of containment to albumen enzymatic synthesis, so
Yield is lower.
(2) cell density measures: in the in-depth cultivation period of embodiment 2,3,4,5,7 and 8.Density measurement: with 721 types point
Light photometer measures A at 600nm wavelength600Value, OD value is in a linear relationship with dry cell weight, the A of a unit600With dry
Bacterium 0.4g/L meter, it is primary per measurement for 24 hours.As a result and statistical analysis is as shown in Fig. 1.
Fig. 1 is influence schematic diagram of the different breeding methods to Shewanella cell density.As seen from the figure, with the cultivation time
Increase, cell density totally shows a increasing trend, but embodiment 2 causes part thalli growth to become feeble and die since environment early period changes, bacterium
Volume density increase is relatively slow, and mid-term thallus increases comparatively fast after adapting to, and the later period tends to be steady；The trend of embodiment 3 is basic with embodiment 2
Unanimously, but compared with 2 growth trend of embodiment it becomes apparent from, is since the change of ingredient in replenishers reduces metabolite to thalli growth
The inhibition of breeding increases thallus proliferative speed；It is since unassembled amino acid makes that the cell density of embodiment 4, which increasess slowly,
Thallus stability in extraneous environmental catastrophe is broken, and decline amount is larger；Embodiment 5 fluctuates greatly compared with embodiment 2, is due to crosslinking
Agent changes the interception for causing amino acid crosslinks structure to absorb energy to thallus；Embodiment 7 is due to being constantly in low carbon, low nitrogen
Under environment, thalli growth breeding is slow；Embodiment 8 is then the swift and violent growth and breeding under high-carbon nitrogen environment early period, in carbon nitrogen content
Thallus breeding tends to be steady after reduction.Therefore, in order to obtain high yield of enzyme, high enzymatic activity, the breeding method of embodiment 2 is more worth
It promotes.
Test example 2:
The optimal reactive temperature and heat stability testing of alkali protease obtained by Shewanella
(1) optimal reactive temperature: 2 gained alkali protease of embodiment is respectively placed in 30,35,40,45,50,55,60,
At 65 DEG C, its enzyme activity is measured.On the basis of taking highest enzyme activity, the enzyme activity at a temperature of other calculates opposite enzyme with respect to highest enzyme activity
It is living, and analyze and do figure, as a result as shown in Figure 2.
Fig. 2 is alkali protease optimal reactive temperature test result schematic diagram.As seen from the figure, the alkali protease is in 30-
There is higher enzyme activity at 50 DEG C, and the enzyme activity highest at 40 DEG C.
(2) heat stability testing: 2,3,5,6 gained alkali protease of embodiment is placed in the borax-NaOH that pH is 13 and is delayed
In fliud flushing system, 1h is kept the temperature at 15,20,25,30,35,40,45,50,55,60 DEG C respectively, is surveyed according to enzyme activity determination method
Fixed residue enzyme activity, saving fresh enzyme solution with 4 DEG C is control, calculates remnant enzyme activity, and analyze and do figure, as a result as shown in Figure 3,4.
Fig. 3 is that the thermal stability of alkali protease changes schematic diagram.As seen from the figure, embodiment 2 and 3 is protected at 15-50 DEG C
Warm 1h, remnant enzyme activity illustrate that protease is still able to maintain higher enzymatic activity between 15-25 DEG C of low temperature, extend 75% or more
The temperature use scope of enzyme.
Fig. 4 is the enzyme activity change curve of alkali protease at low ambient temperatures.As seen from the figure, embodiment 2 and 3 is at 15 DEG C
Lower heat preservation 1h, remnant enzyme activity illustrates that protease is still able to maintain higher enzymatic activity at 15 DEG C still 75% or more, and embodiment 5
Remnant enzyme activity with 6 significantly reduces, and illustrates the adaptability at low ambient temperatures of protease obtained by the breeding method of embodiment 5 and 6
It is poor, and the breeding method of embodiment 2 and 3 can make alkali protease keep high enzyme living at low ambient temperatures, have more aobvious
The beneficial effect of work.
Test example 3:
The optimal pH and pH stability test of alkali protease obtained by Shewanella
(1) optimal pH: it is the slow containing casein solution of 6-14 that 2 gained alkali protease of embodiment, which is respectively placed in pH,
In fliud flushing, the enzyme activity under different pH value is measured.On the basis of taking highest enzyme activity, the enzyme activity under other pH is calculated with respect to highest enzyme activity
Opposite enzyme activity, and analyze and do figure, as a result as shown in Figure 5.
Fig. 5 is alkali protease optimal pH test result schematic diagram.As seen from the figure, the alkali protease is when pH is 8-14
There is a higher enzyme activity, and the enzyme activity highest at 13.
(2) embodiment 2,6 gained alkali proteases pH stability test: are placed in the buffer solution system that temperature is 40 DEG C
In, 1h is kept the temperature in the case where pH is 8-14 respectively, remaining enzyme activity is measured according to enzyme activity determination method, saves fresh enzyme solution with 4 DEG C
For control, remnant enzyme activity is calculated, and analyzes and does figure, as a result as shown in Figure 6.
Fig. 6 is the pH stability change schematic diagram of alkali protease.As seen from the figure, the alkali protease of embodiment 2 is in pH
When to keep the temperature 1h in the environment of 8-14, remnant enzyme activity is 88% or more, and 6 gained protease of embodiment is higher than 12 environment in pH
Under, enzyme activity significantly reduces, and illustrates that breeding method can make alkali protease keep high enzyme under strong alkali environment in embodiment 2
It is living, the pH use scope of enzyme is extended, there is significant benefit.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (10)
1. the breeding method of the Shewanella of high yield alkali protein, including, obtain the Shewanella bacterium of amino acid assembling modification
Strain；With, make the bacterial strain carry out intermittent low carbon, low nitrogen fermentation in-depth cultivate；
The intermittent low carbon, low nitrogen fermentation is by in-depth cultivation period, at least one predetermined time to cultivating predetermined amount
Add replenishers in the cultivation system of time, at the same secondary cryogenic strong alkali environment and realize.
2. the breeding method of the Shewanella of high yield alkali protein according to claim 1, it is characterised in that: the alkali
The preparation step of the Shewanella bacterial strain of acidic amino acid assembling modification are as follows: amino acid is dispersed in the electrolyte that pH is 8-9
In solution, Shewanella bacterial strain is then added with the ratio of 10-20g/L, then add crosslinking agent thereto, then carries out shaking table shake
Swing reaction 1-2h to obtain the final product.
3. the breeding method of the Shewanella of high yield alkali protein according to claim 2, it is characterised in that: the ammonia
Base acid is basic amino acids lysine or arginine；Concentration of the amino acid in mixed system is 2-5wt%.
4. the breeding method of the Shewanella of high yield alkali protein according to claim 2, it is characterised in that: the friendship
Join the 0.5-1.5% that agent additive amount is bacterial strain weight；The crosslinking agent includes 1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine,
Wherein the weight ratio of 1,2- ethylenediamine, 5- chloro pentane acid and phosphatic hydroxylamine is 2:0.3-0.8:0.1-0.5.
5. the breeding method of the Shewanella of high yield alkali protein according to claim 1, it is characterised in that: the depth
Change and cultivate operation are as follows: cultivation period 7-10d wherein adding replenishers into cultivation system every 20-24h, while will be cultivated
Environment by pH is 6.5-8, temperature is 25-40 DEG C and is adjusted to low temperature strong alkali environment, continues after cultivating 3-6h, nurturing an environment is adjusted
Continue to cultivate for normal nurturing an environment, with the adjusting of nurturing an environment when adding replenishers every time, until cultivation period terminates.
6. the breeding method of the Shewanella of high yield alkali protein according to claim 1 or 5, it is characterised in that: institute
State low temperature strong alkali environment operating condition are as follows: temperature is 15-25 DEG C, pH 9-12.
7. the breeding method of the Shewanella of high yield alkali protein according to claim 1 or 5, it is characterised in that: institute
The single use amount for stating replenishers is the 0.4-1% of culture solution weight；Contain sucrose, casein and chlorination in the replenishers
Ammonium, weight ratio 1:0.5-1:0.2-0.5.
8. the breeding method of the Shewanella of high yield alkali protein according to claim 1, it is characterised in that: the training
Educating method further includes stabilizing to cultivate；The step of stabilisation is cultivated are as follows: the microorganism collection after cultivating in-depth, and access
In 2216E sea water medium, Yu Wendu 20-45 DEG C, pH stabilize cultivation 2-3d under conditions of being 8-12 after, it is centrifuged and simultaneously receives respectively
Collection cultivates gained thallus and liquid.
9. the described in any item methods of claim 1-8 cultivate purposes of the resulting Shewanella in alkali protease production.
10. purposes of the Shewanella according to claim 9 in alkali protease production, it is characterised in that: the alkali
Property protease applicable pH be 8-14, Applicable temperature be 15-50 DEG C, belong to the low temperature resistant albuminoid of resistance to highly basic enzyme.
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| CN111440807A (en) * | 2020-04-07 | 2020-07-24 | 上海海洋大学 | Shewanella WP3 mutant strain with high yield of low-temperature catalase as well as construction method and application thereof |
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