CN1289659C - High temperature neutral protenase strains, high temperature neutral proleinase and process thereof - Google Patents
High temperature neutral protenase strains, high temperature neutral proleinase and process thereof Download PDFInfo
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Abstract
The present invention discloses a bacillus licheniformis strain with high yield for generating high temperature neutral protenase and a technology for producing high temperature neutral protenase by the bacillus licheniformis strain. The present invention simultaneously provides high temperature neutral protenase and an enzyme preparation. The high temperature neutral protenase of the present invention has the characteristics of good thermal stability and high enzyme activity. The present invention uses the bacillus licheniformis strain for establishing the technical skill of a system of strain storage, rejuvenation and selection breeding. A fermentation period is shortened by the optimization screening of a culture medium recipe and the optimization control of a fermentation technical condition for establishing a stable industrial fermentation technology. Moreover, the new technology for the pretreatment, the concentration and the spray drying of fermentation liquor is established. The present invention enhances and stabilizes the quality of the enzyme preparation, reduces environmental pollution, reduces the cost by more than 40% and makes the quality of the preparation achieve the high quality standard of industry. The present invention is widely applied to each field of national economy.
Description
Invention field
The present invention relates to microorganism and microbial fermentation field.Specifically, the present invention relates to a kind of lichem bacillus strain, high temperature neutral protease and production technique and zymin that produces the high temperature neutral protease.
Background technology
Proteolytic enzyme is the biological catalyst that a class is had many uses.Conventional neutral protease is found the earliest and is applied to be produced, and has important industrial value, and its product is used widely in multiple industries such as leather, weaving, chemical industry, food, medicine.Because its thermo-labile, unstable, thereby its suitability for industrialized production and widespread use have been limited.Bacillus subtilis neutral proteinase is pH7.0,60 ℃ of following processing 15 minutes, and enzyme is lived loss more than 90%; Aspergillus terricola 3942 neutral proteases are pH7.5,55 ℃ of following processing 10 minutes, and its enzyme deactivation is more than 80%; The thermostability of actinomycetes 166 neutral proteases is poorer, and only more stable below 35 ℃, 45 ℃ can very fast inactivation.
Sixties Mo, American-European all states added proteolytic enzyme in the washing composition, promoted the once big change of detergent industry, the seventies is along with bionic rise and development, expanded the frontier of nucleic acid and protein engineering, risen the novel industry of toolenzyme, and biotechnology is used to transform the generation bacterium of enzyme, has then further promoted enzymology and zymin industrial expansion, so enzyme has become the central topic of relevant international academic conference.Because Sumizyme MP and the desired reaction conditions of aspartic protease have limited its widespread use,, carry out research both at home and abroad in succession, but all be confined to the normal temperature type neutral protease neutral protease from early eighties.And the poor heat stability of normal temperature neutral protease, production and application art technical sophistication, cost height.In order to solve this deficiency, begin all to be devoted in the world research from the nineties, and make certain gains high temperature neutral protein enzyme-producing bacteria and condition of enzyme production.External pertinent literature mainly is the research of relevant high temperature neutral protease and genetic engineering bacterium thereof, the zymogenic bacteria kind that it relates to has: B.brevis MIB001 (bacillus brevis), B.spYP-T (Bacillus subtilus), Pseudomonas aurantiacus J-10-f1 (pseudomonas), B.stearothermophilus (bacstearothermophilus) HY-69, Chloroflexus aurantiacus J-10-f1, B.stearothermophilus MK232, B.stearothermophilus 313-1, EAI etc., the isolated Pseudomonasaeruginosa CCRC15541 in Taiwan wherein, the high temperature neutral protease of its generation is the metal enzyme, 60 ℃ of optimal reactive temperatures, pH7.0,1 hour enzyme of effect is lived and is still kept 85% under 60 ℃; The outer high temperature neutral protease of a kind of born of the same parents that B.stearothermophilus produces, 60 ℃ of optimal reactive temperatures, optimal pH 6.0,30 minutes enzymes of effect are lived and are preserved 90% under 90 ℃; B.stearothermophilus high temperature neutral protease gene is expressed in B.subtilis, make output improve 29 times, the proteolytic enzyme pH neutrality of generation, 75 ℃ of optimal reactive temperatures act on 30 minutes down at 80 ℃ and can keep active 85%.Industrialization Progress is comparatively slow, and output is very low.
At present external high temperature neutral protease research mainly concentrates on the structure of molecular biology and genetic engineering bacterium, the domestic seed selection that mainly concentrates on bacterial classification, the molecular biological characteristic of enzyme, the research of aspects such as the immobilization technology of enzyme and general neutral protease fermentation technique, what relate to the research of high temperature neutral protease is Institute of Microorganism, Academia Sinica, this institute has carried out the research of bacstearothermophilus HY-69 high temperature neutral protease enzymatic property, this enzyme optimal pH 7.5,85 ℃ of optimal reactive temperatures, the enzyme transformation period alive is 22 minutes in the time of 90 ℃, 3 hours enzymes of 80 ℃ of insulations are lived and are still kept 60%, and the report of industrialization aspect is not arranged so far.In addition, the Research on Thermal Stability that improves neutral protease by the immobilization technology of enzyme is also arranged both at home and abroad, but this method can change the pH sphere of action of enzyme.
Summary of the invention
Low at normal temperature neutral protein enzyme reaction temperature, the shortcoming of poor heat stability, the present invention is according to the biological principle that adapts to environmental facies, in the Xinjiang Turfan Prefecture high salinity soil of extremely arid, extremely sweltering heat, isolate a collection of high temperature resistant, anti-saline and alkaline microorganism strains, therefrom filter out a strain and be numbered the bacterial strain of XJT9503, thereby provide a kind of high temperature neutral protease bacterial strain, it has the good characteristic that high yield is produced the high temperature neutral protease, identify through microbiology, be decided to be Bacillus licheniformis (Bacilluslicheniformis).
Simultaneously, on the basis of Bacillus licheniformis of the present invention, the invention provides a kind of production technique of high temperature neutral protease, this technology has possessed technical scale throughput.
The zymin that the present invention also provides a kind of high temperature neutral protease and contained it.
The invention provides a kind of high temperature neutral protease bacterial strain, called after XJT9503, it can produce the high temperature neutral protease with high yield.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100080.Preservation date is on October 14th, 2002, and preserving number is CGMCCNo.0800.This bacterium can utilize glucose to produce acid, also can utilize L-arabinose, the D-seminose, and the D-wood sugar, hydrolyzed starch, liquefy gelatin decomposes casein, can utilize the Citrate trianion growth, edwardsiella hoshinae, and can under 15% sodium-chlor high salinity, grow.Can be than growth in 25 ℃-55 ℃ of the large-temperature ranges, optimum growth temperature is 45 ℃, and growth pH scope is 6-10, and the suitableeest growth scope is 6-8.According to uncle Jie Shi systematic bacteriology handbook second volume, the XJT9503 bacterium is the member in the bacillus, is decided to be Bacillus licheniformis (Bacillus licheniformis).The present invention further sets up the system process technology of culture presevation, rejuvenation and seed selection.
(1) culture presevation: bacterial classification has carried out the preservation test under mineral oil and vacuum freezing condition.Its effect is as described in Table 1:
The different method for preserving of table 1 are to the influence of bacterial strain survival rate
| Treatment process | 7 days | 1 month | 6 months |
| Mineral oil | 99％ | 98％ | 80％ |
| The vacuum preservation | 99％ | 99％ | 99％ |
(2) actication of culture: go bail for and deposit bacterial strain, scrape with inoculating needle and get or picking is connected on the solid LB on a small quantity, after cultivating certain hour under the temperature that is fit to strain growth, activation and is carried out the mensuration of strain enzyme-producing ability for several times continuously.
(3) rejuvenation of spawn: in pilot scale, determined the flow process that a cover keeps producing enzyme stability of characteristics bacterial strain screening.
The bacterium that inoculation activation is good in liquid LB and solid LB and cultivating detects this bacterium characteristic successively, chooses strain excellent and is inoculated in and cultivates on the slant medium, and this bacterial strain bottle is carried out fermenting experiment with shaking, to detect its enzymatic productivity.After detecting its enzymatic productivity and degenerating, this bacterial strain can be used as to be produced bacterial strain and uses.
The invention provides a kind of production technique of high temperature neutral protease, it comprises:
A: (CGMCC.No.0800) carries out the strain activation and culture step to Bacillus licheniformis;
B: the activated spawn of utilizing steps A to obtain is carried out fermentation step;
C: the extraction step after the fermentation.
In the fermenting process, the condition when selecting high temperature neutral protein production of enzyme high is an optimum condition.The about 1%-15% of preferred fermentation inoculum size, the about 42-49 of temperature ℃, air flow about 1: 0.25-0.6vvm, stirring velocity is about 100-350rpm, and fermenting enzyme work reaches more than the 6000u/ml, when reducing sugar 0.5% is following, fermentation ends, generalized case, fermentation period 15-32 hour.
On ferment tank technology, can adopt conventional single batch fermentation technology, also can further adopt stream to add technology.In an embodiment of the invention, adopt conventional single batch fermentation can make fermenting enzyme work reach 8000u/ml, in yet another embodiment of the present invention, after adopting stream to add technology, regulate and control to make the work of single batch fermentation enzyme to bring up to more than the 11000u/ml to the metabolism of carbon source in the fermenting process and nitrogenous source.
During the fermentation, substratum is the basis of zymotechnique.The present invention utilizes shake flask fermentation that fermention medium is screened and optimize, and bacterial activity is detected in experiment.In line with high yield, principle cheaply, on breadboard basis, carried out the substratum screening of industrialization pilot scale research and optimized.Bacillus licheniformis of the present invention (CGMCC.No.0800) there is no special regulation to the nutrition source of substratum, can make to contain the conventional carbon source that is used for microorganism culturing, nitrogenous source in the substratum.For example, the XJT9503 bacterial strain can glucose, maltose, Semen Maydis powder, starch etc. are made carbon source, and enzyme is also produced in growth.Wherein, be that the carbon source thalli growth is the fastest with glucose, but use Semen Maydis powder can make the yield of enzyme maximum of this bacterial strain.Can utilize ammoniacal liquor, peptone, soybean cake powder, corn steep liquor etc. to use as nitrogenous source, but soybean cake powder is an optimum nitrogen source, it obtains, uses easily, and can make the production bacterial strain reach maximum yield of enzyme.
Prove that by experiment metal ion has certain effect to fermentation, Cu ion, Al ion, Fe ion pair are produced enzyme and are had different restraining effect, Na, Zn ion pair product enzyme do not have remarkable effect, and Ca, Mg ion pair produce enzyme and have promoter action, and the K ion can make thalli growth accelerate, and is of value to the product enzyme.Ca, Mg and the K ion of preferred 0.01%-1%.
During the fermentation, the material sterilization all can make fermented liquid pH have greatly changed with bacterial metabolism, and therefore, the pH that the present invention uses phosphoric acid salt to regulate fermenting process in fermention medium changes, and the pH that uses carbonate control material sterilization process to bring changes.
The fermented liquid that produces in the present invention's fermentation makes fermentation broth viscosity higher owing to reasons such as metabolic by-prodss in material properties and the fermenting process, and this causes difficulty for the extraction of enzyme.Therefore fermenting enzyme liquid is removed solid substance, reduce viscosity, just become the difficult point of extraction process.According to the character of fermented liquid and flocculation agent, the present invention is by a large amount of experiments, by the throwing out of flocculation agent, solved the viscosity problem of fermented liquid, and the floss that flocculation agent produced can be removed by the substep centrifuging, obtain clarifying fermented liquid, and the fermentation broth enzyme vigor does not lose substantially.The present invention preferably adopts sodium alginate to handle fermented liquid.More preferably adopt the 0.1%-1% sodium alginate to handle fermented liquid.
Fermented liquid can obtain clarifying fermentation treatment fluid through after the pre-treatment, but it is bigger to handle liquid measure, if fermentation treatment fluid is concentrated, then can reduce the consumption of raw material in the downstream extraction technique and handle liquid measure, reduce production cost, in the preparation of liquid enzymes, reduced memory space simultaneously in a large number.Therefore the present invention studies the concentrated of fermented liquid, and the experiment by a large amount of obtains preferred embodiment.Use the film rotary concentrator to carry out the mother liquor heating and concentrate experiment, under the certain temperature condition of negative pressure, can make fermentation treatment fluid concentrate several times, its enzyme activity does not lose substantially.Use ultrafilter that mother liquor has been carried out molecular retention, respond well.Extraction process of the present invention and without particular limitation can be with an organic solvent and method such as neutral salt precipitation, preferably adopts the method for ammonium sulfate precipitation.The present invention can also add auxiliary material, the disposable dry powder zymin that makes of spray-dried tower in the fermentation treatment fluid after concentrating.The resulting enzyme powder preparation of spraying drying is khaki, does not have caking, no deliquescence phenomenon, and powder is tiny.The fermentation treatment fluid concentration technique is engaged with spraying drying, can produce the enzyme preparation product of different size as required.The present invention can also add sanitas in the fermentation treatment fluid after concentrating, low temperature seal obtains liquid enzyme formulation.
The present invention provides simultaneously and has utilized high temperature neutral protease bacterial strain (CGMCC.No.0800), and the high temperature neutral protease of acquisition not only has general neutral protease characteristic, and it is low to have overcome normal temperature neutral protein enzyme reaction temperature, the shortcoming of poor heat stability.High temperature neutral protease provided by the invention has good thermostability, enzyme is alive high, can make the application of temperature of neutral protease improve 20 ℃, and product has possessed technical scale throughput.Those of ordinary skills are known, and the temperature of reaction of high temperature neutral protease finger protein enzyme is higher, general temperature of reaction 50-70 ℃, react general pH 5.0-8.0 under neutrallty condition.High temperature neutral protease of the present invention is a meta-bolites of producing bacterial strain (CGMCC.No.0800), by optimization to fermention medium and fermentation parameter, enrichment high temperature neutral protease molecule in fermentation liquid, and utilize the physico-chemical property of enzyme molecule to carry out the product separation.High temperature neutral protease of the present invention, optimal reactive temperature are 65 ℃, the suitableeest action pH 7.0-7.2; Molecular weight: enzyme SDS-PAGE detected through gel electrophoresis, to compare with standard protein, determining molecular weight is 40000; Iso-electric point: enzyme is measured through the polyacrylamide isoelectric focusing electrophoresis, and the iso-electric point of enzyme molecule is 6.8; Metal ion is to the effect of enzyme: Ca ion and Mg ions enzyme molecule have activation, and the activity of Cu ion, Fe ion, Al ion and Zn ions enzyme molecule has restraining effect in various degree; The transformation period of enzyme: 50 ℃ of insulations 1 hour, enzyme activity descended 10%, in 45 minutes transformation period of 60 ℃ of following enzymes.
The present invention makes enzyme solid powder preparation and liquid preparation in the pilot scale stage, and quality product meets national relevant industries standard Q/GB1805.3-93.Wherein, the solid zymin is khaki, does not have caking, no deliquescence phenomenon, and the enzyme total recovery is greater than 60%; Every gram enzyme activity is about 5-21.8 ten thousand units; Preserved about 210-575 days.
The solid powder enzyme preparation product of the 50000u/g-150000u/g that the present invention obtains, the yield of its fermentation treatment fluid can reach 70%; The present invention has obtained the liquid enzyme formulation of 30000u/g-100000u/g, and the liquid enzymes yield can reach 90-94%.
At present, the present invention has finished pilot experiment, and identifies examination by the Committee of Experts of the Department of Science and Technology " Chinese biological engineering center ".Adopt national relevant industries standard GB1805.3-93 to detect.Carried out the fermentation pilot scale of 35 batches of 250 liters of fermentor tanks, average enzyme work is more than 8000u/ml; Carried out the fermentation pilot scale of 3 tons of fermentor tanks 11 batches wherein continuous 8 batches, enzyme work is stabilized in more than the 11000u/ml, and the highest fermenting enzyme work reaches 11970u/ml; Under the on-the-spot supervision of two experts of the Department of Science and Technology " Chinese biological engineering center ", carried out 10 tons of fermentation pilot scales that fermentor tank is continuous 2 batches, enzyme work is stabilized in more than the 14000u/ml, and the highest fermenting enzyme work reaches 14934u/ml.Made solid enzyme powder preparation and liquid enzyme formulation, solid zymin yield is more than 60%, and liquid enzymes can reach 90-94%.Its preservation effect is as described in table 2, table 3 and the table 4:
Table 2 liquid enzymes preservation effect:
| Time (my god) | 0 day | 10 days | 40 days | 105 days |
| Enzyme (u/ml) alive | 3096 | 2944 | 3008 | 2568 |
| Rate of loss (%) | 0 | 4.9 | 2.8 | 17 |
Table 3 food grade solid zymin preservation effect:
| Time (my god) | 0 | 30 | 60 | 180 | 210 |
| Enzyme (u/g) alive | 111230 | 111230 | 111230 | 111230 | 109005 |
| Rate of loss (%) | 0 | 0 | 0 | 0 | 2 |
Table 4 technical grade solid zymin preservation effect:
| Time (my god) | 0 | 30 | 60 | 180 | 210 |
| Enzyme (u/g) alive | 153230 | 153200 | 150165 | 145569 | 140972 |
| Rate of loss (%) | 0 | 0 | 2 | 5 | 8 |
Beneficial effect:
A, directed screening through being accredited as Bacillus licheniformis (Bacillus licheniformis), and set up the system process technology of culture presevation, rejuvenation and seed selection to XJT9503 high temperature neutral protease superior strain.The high temperature neutral protease that utilizes strain fermentation of the present invention to obtain has the temperature of reaction height, the characteristics of Heat stability is good.
B, pass through the preferred embodiment of the invention, the for example optimization of culture medium prescription and technological condition for fermentation, establish XJT9503 and produced the stable zymotechnique of bacterial strain, fermentation period was reduced to about 16 hours by 36 hours, the work of 3 tons of fermentor tank enzymes is stabilized in more than the 11000u/ml, and the highest fermenting enzyme work reaches 11970u/ml; The work of 10 tons of fermentor tank enzymes is stabilized in more than the 14000u/ml, and the highest fermenting enzyme work reaches 14934u/ml, surpasses the original plan index (10000u/ml), and cost reduces more than 30% than planned target.
C, the XJT9503 high temperature neutral protease fermentation liquor pretreatment of utilize setting up, concentrate, complete extraction process such as spraying drying, the zymin quality of producing reaches industry gold standard promulgated by the ministries or commissions of the Central Government.Reduced environmental pollution, reduced cost.
Description of drawings
Fig. 1 shows high temperature neutral protease enzyme activity determination method flow diagram of the present invention.
Fig. 2 shows fermentation schema 1.
Fig. 3 shows fermentation schema 2.
Embodiment
Embodiment 1 the present invention selects the preservation and the rejuvenation of bacterial classification for use
Obtained a plant height temperature neutral protease in the Turfan Prefecture separation and produced bacterium XJT9503, through being accredited as genus bacillus (CGMCC No.0800), 65 ℃ of the high temperature neutral protease optimal reactive temperatures that is produced, about 45 minutes of the transformation period of 60 ℃ of following enzymes.
Adopt two kinds of methods to carry out culture presevation, i.e. whiteruss preserving process and vacuum freeze-drying method.The former is used for the bacterial classification short-term and preserves (3-6 month), and the latter is used to produce the long-term preservation (6 months-several years) of bacterial classification.
Preserve the activation of bacterial classification: go bail for and deposit bacterial strain, be equipped with in the test tube of 2 ml sterile waters, vibrated 10 minutes with inoculating needle picking bacterial classification inoculation.Get 0.2 milliliter with aseptic straw then and be added to slant medium (extractum carnis, 3g; Peptone, 10g; Sodium-chlor, 5g; Agar, 15-20g; Water, 1000ml; PH:7.0-7.2) on the plate culture medium, smoothen with glass spatula again, put to cultivate after 12 hours in 46 ℃ of incubators and take out culture medium flat plate.
The screening of superior strain: will activate good bacterial classification inoculation in liquid LB substratum, in 46 ℃, 150rpm cultivated 12 hours down, is that 10-12 coats on the solid LB culture medium flat plate with sterilized water with its dilution, cultivate down in 46 ℃, picking list colony inoculation is on solid LB screening culture medium again, incubated overnight, detect its bacterial plaque and bacterium colony directly than size, what ratio was big is superior strain, it is inoculated on the slant medium (the same), cultivated 12 hours for 46 ℃, preserve down in 4 ℃, simultaneously this bacterial strain is carried out fermenting experiment with shaking bottle, to detect its enzymatic productivity (as follows).Detecting its enzymatic productivity degeneration person is not taken place can be used as and produce bacterial strain and use.
1) XJT9503 high temperature neutral protease enzyme activity detection method
---issue in 1993-07-29 according to Ministry of Light Industry of the People's Republic of China (PRC), and in " People's Republic of China's industry standard " QB/T 1803,1804-93 of 1994-03-01 enforcement, QB/T1805,1806-93, the general test method of industrial enzyme preparation, and XJT9503 high temperature neutral protease characteristic, to be decided to be 65 ℃, pH be 7.2 to temperature in test process.
(1) definition
1 gram solid enzyme powder (or 1ml liquid enzymes), at a certain temperature with the pH condition under, it is an enzyme activity unit that 1 minute hydrolyzed casein produces 1ug tyrosine, represents with u/g (u/ml).
(2) folin's methods
2.1 principle
XJT9503 high temperature neutral protease is at 65 ℃, and under the pH7.2 condition, the hydrolyzed casein substrate produces the amino acid that contains phenolic group, under alkaline condition, with Folin reagent (Folin) reduction, generates molybdenum blue and tungsten blue, uses spectrophotometric determination, calculates its enzyme activity.
2.2 reagent and solution
2.2.1 the preparation of Folin reagent
In 2000L ground reflux, add sodium wolframate (Na
2WO
42H
2O) 100g, Sodium orthomolybdate (Na
2MoO
42H
2O) 25g, water 700ml, 85% phosphoric acid 50ml, concentrated hydrochloric acid 100ml, little fiery boiling reflux 10 hours takes off reflux cooler, adds Lithium Sulphate (Li in stink cupboard
2SO
4) 50g, water 50ml and several dense bromine waters (99%), little again boiling 15 minutes, to remove unnecessary and bromine (still having green need add bromine water again after cold), cooling adds water and is settled to 1000ml.Mixing filters.It is golden yellow that the reagent that makes should be, and stores in the brown bottle.
Use solution: a Folin reagent mixes with two parts of water, shakes up.
2.2.2 sodium carbonate solution c (Na
2CO
3)=0.4mol/L
Take by weighing anhydrous sodium carbonate (Na
2CO
3) 42.4g, with water dissolution and fixed molten to 1000ml.
2.2.3 trichoroacetic acid(TCA) c (CCl
3COOH)=0.4mol/L
Take by weighing trichoroacetic acid(TCA) (CCl
3COOH) 65.4g is with water dissolution and fixed molten to 1000ml.
2.2.40.02M, the preparation of pH7.2 phosphoric acid buffer
Take by weighing Sodium phosphate dibasic (Na
2HPO
412H
2O) 4.90g and SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
42H
2O) 0.99g is dissolved in water and fixed molten to 1000ml.
2.2.550g/L casein solution
Take by weighing casein 1.000g, be accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution moistening after, the about 80ml of phosphoric acid buffer that adds an amount of pH7.2, in boiling water bath, heat while stirring, until dissolving fully, after the cooling, change in the 100ml volumetric flask, be diluted to scale with an amount of pH buffered soln.This solution is preserved in refrigerator, and validity period is three days.
2.2.6100ug/ml L-tyrosine standardized solution
A. take by weighing the L-tyrosine 0.1000g of constant weight, be accurate to 0.0002g, fixed molten with 1mol/L hydrochloric acid 60ml dissolving back to 100ml, be 1mg/ml tyrosine standardized solution.
B. get 1mg/ml tyrosine standardized solution 10.00ml, molten with 0.1mol/L salt acid cut to 100ml, promptly obtain 100ug/ml
2.3 instrument and equipment
2.3.1 65 ± 0.2 ℃, 40 ± 0.2 ℃ of waters bath with thermostatic control.
2.3.2 spectrophotometer should meet the regulation of GB 9721.
2.2.4 determination step
2.2.4.1 the drafting of typical curve
A.L-tyrosine standardized solution
The according to the form below preparation
| The pipe number | Tyrosine concentration of standard solution ug/ml | Get 100ug/ml tyrosine standardized solution volume ml | The volume ml of water intaking |
| 0 | 0 | 0 | 10 |
| 1 | 10 | 1 | 9 |
| 2 | 20 | 2 | 8 |
| 3 | 30 | 3 | 7 |
| 4 | 40 | 4 | 6 |
| 5 | 50 | 5 | 5 |
B. get above-mentioned each 1.00ml of liquid (must do parallel test) that absorbs respectively, respectively add 0.4mol/ml sodium carbonate solution 5.00ml, Folin reagent 1.00ml, place 40 ℃ of water-bath colour developings 20 minutes, take out with spectrophotometer in wavelength 680nm, the 10mm cuvette, with No. 0 pipe not containing tyrosine is blank, measures its absorbancy respectively.With the absorbance A is ordinate zou, and the concentration c of tyrosine is an X-coordinate, drawing standard curve (this line should pass through zero point).
According to mapping or using regression equation, calculate the amount (ug) of the tyrosine when absorbancy is 1, be extinction constant K value.Its K value should be about 100.
2.2.4.2 the preparation of enzyme sample to be measured and mensuration
A. take by weighing enzyme powder 1-2g, be accurate to 0.0002g (or imbitition enzyme 1.00ml), phosphoric acid buffer dissolving with a small amount of pH7.2, and grind with smashing, then with in the supernatant liquor impouring volumetric flask, add appropriate amount of buffer solution in the sediment again, grind and smash 3-4 time, in last all immigrations volumetric flask, be settled to scale, shake up with damping fluid, with four layers of filtered through gauze, filtrate is diluted to suitable concentration with damping fluid again according to enzyme activity,, dilutes good enzyme liquid and places refrigerator to preserve with (being diluted to the test fluid light absorption value in the 0.25-0.45 scope) for test.
When the enzyme powder is measured, the extension rate reference table:
| Enzyme unit alive | Extension rate | Dilution for the first time | Dilution for the second time |
| 20,000 | 2000 | 2g → 200ml (100 times) | 5ml → 100ml (20 times) |
| 30,000 | 2500 | 2g → 500ml (250 times) | 5ml → 50ml (10 times) |
| 40,000 | 4000 | 2g → 200ml (100 times) | 5ml → 200ml (40 times) |
| 50,000 | 5000 | 2g → 500ml (250 times) | 5ml → 100ml (20 times) |
| 8,100,000 | 10000 | 2g → 500 (250 times) | 5ml → 200ml (40 times) |
B. measure
Earlier casein solution is put into 65 ℃ of waters bath with thermostatic control, preheating 5 minutes.And by as described in Figure 1 procedure operation:
C. calculate
X＝A*K*4/10*N
In the formula: the enzyme activity of X-sample, u/ml (u/g);
The mean light absorbency of A-sample parallel test;
K-extinction constant;
The cumulative volume of 4-reaction reagent, ml;
10 minutes 10-reaction times, in minute;
The N-extension rate.
The result who is measured is expressed as integer.Parallel test misses relatively
Difference is no more than 3%.
The screening of embodiment 2, fermention medium of the present invention
Substratum is the basis of zymotechnique technology, in line with high zymogenic rate, principle cheaply, has carried out the substratum screening of industrialization pilot scale research and optimize on the basis of laboratory study.Carbon source is selected Semen Maydis powder, and nitrogenous source is a soybean cake powder, and adds 0.01%-1%Ca, Mg and K ion, promotes the raising of thalline production and zymogenic rate, and regulates the substratum variation of pH during the fermentation with phosphoric acid salt and carbonate.
1, screening culture medium
| Title | Consumption |
| Extractum carnis | 3g |
| Peptone | 10g |
| Sodium-chlor | 5g |
| Agar | 15-20g |
| Casein | 5g |
| Water | 1000ml |
Annotate: pH7.0-7.2, casein need dissolving fully.
2, shake bottle and detect substratum
| Title | Consumption (%) |
| Glucose | 6 |
| Semen Maydis powder | 3 |
| Sodium phosphate dibasic | 0.4 |
| Sal epsom | 0.02 |
| Calcium chloride | 0.02 |
In 45-46 ℃, 240 rev/mins, 200ml material/bottle (500ML shakes bottle), add tampon, cultivated 60 hours, the centrifugal thalline that goes of 4000rpm, detection fermentation broth enzyme output when production of enzyme reaches 3000u/ml when above, proves that bacterium throughput meets production requirement.
3, seed culture medium and fermention medium
A. seed tank culture base
| Title | Consumption (%) |
| Semen Maydis powder | 2 |
| Soybean cake powder | 1 |
| Sodium phosphate dibasic | 0.4 |
| Sal epsom | 0.02 |
| Calcium chloride | 0.02 |
| Fish meal | 0.4 |
| Defoamer (bubble enemy) | 0.03 |
B. fermentation tank culture medium
| Title | Consumption (%) |
| Semen Maydis powder | 3 |
| Glucose | 0.5 |
| Soybean cake powder | 1.5 |
| Fish meal | 0.2 |
| Sodium phosphate dibasic | 0.4 |
| Sal epsom | 0.02 |
| Calcium chloride | 0.02 |
| Yellow soda ash | 0.085 |
| Defoamer (bubble enemy) | 0.03 |
Embodiment 3, bottle, 7 liters and 25 liters of Study on Fermentation of shaking of the present invention
Find that in shaking bottle (adopting above-mentioned screening culture medium) test shaking speed and triangular flask charge amount have considerable influence to the productive rate of this enzyme, improve shaking speed within the specific limits the productive rate of enzyme is improved; The bottled 150ml substratum of 500ml triangle is adorned the zymogenic rate height of 200ml substratum, and the zymogenic rate of this explanation XJT9503 bacterium becomes positive correlation within the specific limits with dissolved oxygen amount.
Gone up through tens of batches of evidences the dependency of zymogenic rate and dissolved oxygen at 7 liters of automatic fermentor tanks (adopting the above-mentioned bottle that shakes to detect substratum), determined optimization technology, leavening temperature 45-46 ℃, air flow 1: 0.6vvm, stirring velocity is 350rpm, tank pressure is 0.1MPa, fermentation period 32 hours, and zymogenic rate reaches 6000u/ml; On 25 liters of fermentor tanks, optimize the result that technological test has further confirmed 7 liters of fermentor tanks.
The research of embodiment 4,250 liters of automatic ferment tank technologies of the present invention
With reference to accompanying drawing 2 described flow processs, on 250 liters of fermentor tanks, carry out 35 batches and optimize the Technology experimental study, finally grinding fixed technology is inoculum size 5%, 46 ℃ of temperature, air flow 1: 0.45nnm, mixing speed 220rpm, fermentation period 16-18 hour, average enzyme was lived to more than the 8000u/ml.
It is one of effective means that improves yield of fermentation product that 250 liters of fermentor tank streams add technology, can effectively solve the contradiction of thalli growth and meta-bolites in the fermenting process.It is comparatively slow in the metabolism of producing enzyme peak sugar futures that XJT9503 high temperature neutral protease produces bacterium, but directly related with the productive rate of enzyme at the concentration and the biomass that produce enzyme stage sugar.Find in the experiment only to miscarry that to cause thalline be thalli growth by enzymic transformation in the sugaring meeting producing the enzyme stage, be unfavorable for that the enzyme productive rate improves.Do not flow sugaring producing the enzyme stage, stream adds nitrogenous source enzyme productive rate and loses raising only and separately.This is explanation just, needs suitable supplementary carbon source and nitrogenous source in the product enzyme stage.Therefore the present invention changes according to pH during the fermentation, and stream adds a certain amount of sugar and nitrogenous source, and the productive rate of enzyme obviously improves, and reaches more than the 10000u/ml.
The research of embodiment 5,3 tons of ferment tank technologies of the present invention
On the basis of 250 liters of ferment tank technical studies, carry out 3 tons of fermentor tank technical studies, fermention medium and processing condition are further optimized, through batch experiment surplus 10, all achieve success, measure enzyme with the method for embodiment 1 and live, enzyme work is stabilized in 11000u/ml, is up to more than the 13000u/ml.
With reference to the flow process shown in the accompanying drawing 3, the activation inoculation of screening is shaken in the bottle in the 500ml that the 150ml seed culture fluid is housed, place 46 ℃, rotating speed 240rpm cultivates after 16 hours down, inoculum size with 2% is inoculated in the 25L seeding tank, at 46 ℃, rotating speed 100rpm, air flow 1: 0.3vvm cultivates down after 8 hours culture transferring in 300 liters of seeding tanks, culture transferring is in 3 tons of fermentor tanks again, with 46 ℃, rotating speed 180rpm, air flow 1: the 0.25vvm bottom fermentation was cultivated 16-18 hour, measuring the fermenting enzyme productive rate reaches more than the 10000u/ml, residual sugar is below 0.5%, and produces gemma in a large number and can play jar.The XJT9503 that the present invention establishes produces the stable zymotechnique of bacterial strain, and the work of 3 tons of fermentor tank enzymes is stabilized in more than the 11000u/ml, and the highest fermenting enzyme work reaches 11970u/ml; The work of 10 tons of fermentor tank enzymes is stabilized in more than the 14000u/ml, and the highest fermenting enzyme work reaches 14934u/ml, surpasses the original plan index (10000u/ml), and cost reduces more than 30% than planned target.
Embodiment 6, Study on extraction of the present invention
1) fermentation liquor pretreatment technology of the present invention
Fermented liquid of the present invention makes fermentation broth viscosity higher owing to reasons such as metabolic by-prodss in material properties and the fermenting process, and this causes difficulty for the extraction of enzyme.Therefore fermenting enzyme liquid is removed solid substance, reduce viscosity, just become the difficult point of extraction process.The contriver once taked methods such as 10000rpm high speed centrifugation, organic solvent, high salt to handle respectively, did not all obtain desired result, was not that enzyme liquid opacity can not be fallen, and be exactly that the enzyme activity loss is bigger, or cost was higher.In the selection of flocculation agent with use breakthrough has been arranged, adopted sodium alginate to handle fermented liquid and obtained desired result afterwards.The formed condensation product of sodium alginate obtains clarifying enzyme liquid by the centrifugal removal of substep.The present invention can adopt about 0.1%-1% sodium alginate to handle fermented liquid.
2) enzyme liquid concentration technique of the present invention
It is the key technology that reduces cost that the present invention concentrates through pretreatment secondary fermentation enzyme liquid.The present invention adopts the film vacuum concentration, about 40 ℃, approximately-0.094～-0.096Mpa vacuum tightness under, pretreatment fluid can be concentrated 2-3 doubly in 45-55 minute, the loss of living of its enzyme is no more than 10%.
3) preparation of high temperature neutral protease of the present invention and zymin
The present invention has done organic solvent and the sedimentary test of neutral salt, and it is best drawing with ammonium sulfate precipitation.Through pre-treatment and spissated enzyme liquid, utilize the ammonium sulfate precipitation of 25% saturation ratio to remove a large amount of foreign proteins, centrifugal back re-adjustment ammonium sulfate saturation ratio to 55%, and add 1% diatomite filtration, air-dry.The total recovery of enzyme can reach more than 70%.Another kind of preparation method is that through pre-treatment and spissated enzyme liquid adding 7%-9% sodium-chlor and 1%-2% Zulkovsky starch etc., spraying drying directly obtains dried enzyme powder preparation again.The enzyme powder is khaki, does not have caking, no deliquescence phenomenon.Enzyme total recovery about 68%.Prepared 50,000 u/ gram-21.8u/ gram zymin reaches industry gold standard promulgated by the ministries or commissions of the Central Government.Consider that from environmental protection, cost and preservation period this will be the best enzyme preparation technology of large-scale industrial production.
The preparation process of liquid enzyme formulation is simple, and the pretreatment fluid that directly will ferment adds the sanitas low temperature seal and gets final product.
Claims (10)
1, a kind of Bacillus licheniformis CGMCC.No.0800 with generation high temperature neutral protease ability.
2, a kind of production technique of high temperature neutral protease, it comprises:
A: Bacillus licheniformis CGMCC.No.0800 is carried out the strain activation and culture step;
B: the activated spawn of utilizing steps A to obtain is carried out fermentation step;
C: the extraction step after the fermentation.
3, production technique as claimed in claim 2 is characterized in that in the fermentation step, and inoculum size is 1%-15%, leavening temperature 42-49 ℃, and air flow 1:0.25-0.6vvm, fermenting enzyme work reaches more than the 6000u/ml, when reducing sugar 0.5% is following, fermentation ends.
4, production technique as claimed in claim 3 is characterized in that in the substratum adding Ca, Mg and the K ion of 0.01%-1%, and regulates or carbonate is regulated and kept the stable of pH in the fermenting process with phosphoric acid salt.
5, production technique as claimed in claim 2 is characterized in that extraction step comprises the sodium alginate that adopts 0.1%-1% as flocculation agent pre-treatment fermented liquid, and centrifugal removal condensation product obtains clarifying enzyme liquid.
6, production technique as claimed in claim 5, it is characterized in that sodium alginate is handled after, further comprise concentrating enzyme liquid step: adopt the film vacuum method, temperature 35-45 ℃, vacuum tightness 0.094～-0.096Mpa.
7, production technique as claimed in claim 6 is characterized in that adding in the concentrated solution 7%-9% sodium-chlor and 1%-2% Zulkovsky starch, spraying drying again.
8, production technique as claimed in claim 2, strain activation and culture step before it is characterized in that fermenting comprises the bacterial screening step, this screening step comprises that shaking bottle with the superior strain utilization carries out fermenting experiment, detect its enzymatic productivity, degeneration person is not taken place and be can be used as the use of production bacterial strain in its enzymatic productivity.
9, a kind of high temperature neutral protease, it utilizes the described Bacillus licheniformis CGMCC.No.0800 of claim 1, be prepared from by the described production technique of claim 2, and the optimal reactive temperature of this high temperature neutral protease is 65 ℃, molecular weight is 40000, and the iso-electric point of enzyme molecule is 6.8.
10, a kind of solid zymin that contains high temperature neutral protease as claimed in claim 9, it is khaki, does not have caking, no deliquescence phenomenon; In the fermentation step leavening temperature 45-46 ℃; The enzyme total recovery is greater than 60%; Every gram enzyme activity is 5-21.8 ten thousand units; Preserved 210-575 days.
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| CN110408568A (en) * | 2019-08-09 | 2019-11-05 | 中国农业科学院饲料研究所 | The bacillus licheniformis of one plant of high proteinase yield and its enzymatic production method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101117623B (en) * | 2006-08-04 | 2011-01-12 | 新疆农业科学院微生物应用研究所 | Refractory metal prolease gene engineering bacterium and acquiring method therefor |
| CN101215537B (en) * | 2007-12-29 | 2010-06-16 | 北京欣博阳科技有限公司 | Bacillus licheniformis and application thereof |
| CN101215538B (en) * | 2007-12-29 | 2010-06-16 | 北京欣博阳科技有限公司 | Bacillus licheniformis and application thereof |
| CN101555470B (en) * | 2009-04-28 | 2011-04-20 | 陕西省科学院酶工程研究所 | Spray drying method of enzyme preparation zymotic fluid |
| CN103865907B (en) * | 2013-12-13 | 2016-01-20 | 湖南鸿鹰生物科技有限公司 | A kind of neutral protease of heat-flash stability |
| CN103667150B (en) * | 2013-12-13 | 2015-06-24 | 湖南鸿鹰生物科技有限公司 | Bacillus subtilis capable of producing neutral protease with strong heat stability and application of bacillus subtilis |
| CN107201354A (en) * | 2017-07-04 | 2017-09-26 | 北京科为博生物科技有限公司 | A kind of neutral proteinase and its gene and application |
| CN110760466B (en) * | 2019-11-18 | 2021-04-20 | 山东隆科特酶制剂有限公司 | Bacillus subtilis for producing high-temperature-resistant neutral protease and application thereof |
| CN112458071A (en) * | 2020-11-27 | 2021-03-09 | 王雪克 | Production process of high-temperature neutral protease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110408568A (en) * | 2019-08-09 | 2019-11-05 | 中国农业科学院饲料研究所 | The bacillus licheniformis of one plant of high proteinase yield and its enzymatic production method |
| CN110408568B (en) * | 2019-08-09 | 2021-06-08 | 中国农业科学院饲料研究所 | Bacillus licheniformis capable of producing protease in high yield and fermentation enzyme production method thereof |
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