CN106434457B

CN106434457B - One plant of alkali protease superior strain and its produced alkali protease

Info

Publication number: CN106434457B
Application number: CN201610886191.5A
Authority: CN
Inventors: 路福平; 刘逸寒; 李玉
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2016-10-11
Filing date: 2016-10-11
Publication date: 2019-05-10
Anticipated expiration: 2036-10-11
Also published as: CN106434457A

Abstract

The invention belongs to bioengineering fields, and in particular to gram Lloyd's bacillus of one plant of high yield alkali protein and its produced alkali protease.Described gram of Lloyd's bacillus (Bacillus clausii), deposit number are as follows: CGMCC No.12953.Mutagenic obtained high yield alkali protein bacterial strain is to pass through low energy N by gram Lloyd's bacillus in the present invention⁺On the basis of ion implantation mutagenesis, alkali protease superior strain obtained by plasma mutagenesis is carried out again, effectively improve the enzymatic productivity of bacterial strain, the shaking flask vigor maximum value of its alkali protease reaches 79000U/mL, 51.9%, 60T scale fermentation tank enzyme activity, which is improved, compared with starting strain reaches 88000U/mL.

Description

One plant of alkali protease superior strain and its produced alkali protease

Technical field

The invention belongs to bioengineering, are related to induction mutation of bacterium more particularly to a kind of alkali protease superior strain and its institute The alkali protease of production.

Background technique

Alkali protease (Alkaline Protease) refers to aminosal peptide bond under conditions of pH value is alkalinity Enzyme, optimal pH are generally 9~11, belong to the serine protein hydrolase class in endopeptidase, are mainly used in enzyme detergent Industry is also widely used in the industry such as process hides, silk, feed, medicine, food, environmental protection.

Alkali protease is most found early in the pancreas of pig.1914, Germany scientist Rohm and Haas were first by pancreas egg White enzyme and sodium carbonate intermixture are used as washing soaking agent (Burnus).1945, the Dr.Jaag etc. of Switzerland had found micro- life Object alkali protease makes protease be possible to be widely used in detergent industry.1956, the first contained microorganism and produces alkalinity Albumen enzyme detergent enters market.1963, Novo Nordisk Co., Ltd (existing Novozymes Company), which has found, was more suitable for detergent Alkali protease Alcalase, hereafter, enzyme preparation is widely used in detergent product.In subsequent 20 years, bacterium class Protease is the commercialization enzyme preparation for being uniquely applied to detergent.Currently, worldwide protease is in industrial enzyme One kind with the most use accounts for about the 60% of enzyme total amount, and wherein alkali protease just accounts for 25%, before its huge applications in business Scape and the important function in basic research attract international and domestic many companies and research unit and competitively carry out to it in many ways The research in face.Today, Novozyme and Genencor International control the share of global alkali protease 95%.

In recent years, the ion beam mutation source new as one kind is in terms of mutation breeding because of its unique mutagenic mechanism and biology It learns effect and develops extremely rapid, compared with classic mutagenesis source, ion implanting also has dynamic other than with energy sedimentary effect Amount transmitting, quality deposition and charging neutrality and the effects such as exchange, it rolls into one physical mutagenesis and chemical mutagenesis characteristic, can In the case where low dosage injection, the aberration rate of biology is significantly improved, acquisition damage is light, mutation rate is high, the spectrum of mutation is wide, hereditary steady Fixed Mutagenic Effect.Ion implantation technique is widely used in the breeding and Upgrading of microorganism strain excellent, breeding More industrially with the biological new varieties of larger application prospect, and achieve significant economic benefit and social benefit. Meanwhile atmospheric pressure at room plasma mutagenesis (ARTP) is used as a kind of physical mutagenesis means, refers to the plasma using breeding machine Emission source, radiates a branch of plasma, and the exposed sample of direct irradiation has mild condition and quickly thallus can be made to be mutated, grasps The advantages that work is easy, does not need vacuum plant, without side-effects to human body, no pollution to the environment.Plasma is a large amount of phase interactions It but is to be in same layer with solid-state, liquid, gaseous state still in the meta system of the charged particle composition under non-bound state 4th state of the substance on secondary.Plasma is ionized gas, these substances can be with the intracorporal large biological molecule (enzyme or DNA) of biology Interaction, so as to cause the death or mutation of organism.It is one that plasma mutagenesis, which is applied to microorganism mutation breeding, Brand-new technology.

Summary of the invention

Low energy N is used the object of the present invention is to provide a kind of⁺Ion implantation technique and atmospheric pressure at room plasma induced-mutation technique The Bacillus clausii for the high yield alkali protein that mutagenic and breeding obtains, this method effectively improve Bacillus clausii and produce alkali The ability of property protease.

The Bacillus clausii is specially Bacillus clausii (Bacillus clausii) L-7.The bacterial strain is It was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 12nd, 2016, address: court, Beijing No. 3 Institute of Microorganism, Academia Sinica, institute of positive area's North Star West Road 1, postcode 100101, deposit number CGMCC No.12953。

The Bacillus clausii L-7 warp: starting strain screening → N⁺Plate primary dcreening operation after ion implantation mutagenesis → mutagenesis (high yield) → shaking flask secondary screening (high yield) → room temperature chamber pressure plasma mutagenesis → plate primary dcreening operation (high yield) → shaking flask secondary screening (high yield) → Mitotic stability test and etc. screening obtain.

The method of the Bacillus clausii L-7 fermentation production of alkaline protease is as follows:

(1) seed culture: the Bacillus clausii L-7 that one ring of picking is newly cultivated from inclined-plane accesses seed culture fluid In, 200r/min, 34 DEG C of culture 12h；

(2) it fermentation tank culture: transfers according to the inoculum concentration of 5% (v/v) into the fermentor, (fermentation of fermentation tank capacity 70% Culture medium), temperature: 34 DEG C；Revolving speed: 100~600r/min；Ventilation quantity: 1: 0.5~1: 2.5；Dissolved oxygen maintains 20~30%； The hydrochloric acid of auto-feeding ammonium hydroxide and 20% (v/v) in fermentation process, make fermentation liquid pH value maintain 7.0；Fermentation time 50-60h；

The seed culture medium is (g/L): yeast extract 5, tryptone 5, glucose 10, K₂HPO₄18, pH is natural；

The fermentation medium is (g/L): yeast extract 17, cottonseed meal 30, maltodextrin 100, sodium citrate 3, chlorine Change calcium 2.6, K₂HPO₄18, pH is natural.

The produced alkali protease zymologic property of Bacillus clausii L-7 is as follows:

(1) optimum temperature of the alkali protease is 40 DEG C；

(2) the most suitable action pH of the alkali protease is 11；

(3) enzymatic activity of the alkali protease: the maximum value of the shaking flask enzyme activity of alkali protease reaches 79000U/mL, 51.9%, 60T scale fermentation tank enzyme activity, which is improved, compared with starting strain reaches 88000U/mL；

(4) pH stability and thermal stability: in pH6~12,40 DEG C of heat preservation 1h, remnant enzyme activity is 86% or more；40~ 50 DEG C, pH11 heat preservation 1h, remnant enzyme activity have good pH stability and thermal stability 74% or more.

The utility model has the advantages that

1, production alkali protease bacterial strain mutagenic obtained in the present invention is to pass through low energy N by Bacillus clausii⁺From On the basis of son injection mutagenesis, using alkali protease superior strain obtained by atmospheric pressure at room plasma mutagenesis, effectively mention The high enzymatic productivity of bacterial strain, the shake-flask fermentation enzyme activity power maximum value of alkali protease reaches 79000U/mL, compared with starting strain It improves 51.9%, 60T scale fermentation tank enzyme activity and reaches 88000U/mL, production cost can be reduced, obtain good economic effect Benefit.

2, in the present invention, the most suitable action pH of the alkali protease obtained by the strain fermentation screened is 11, most suitable effect Temperature is 40 DEG C, substantially increases the optimal pH of enzyme, increases the use scope of enzyme.

3, for alkali protease of the invention in pH6~12,40 DEG C of heat preservation 1h, remnant enzyme activity is 86% or more；40~50 DEG C, pH11 keep the temperature 1h, remnant enzyme activity has good pH stability and thermal stability 74% or more.

Detailed description of the invention

Fig. 1 is N of the present invention⁺Influence curve of the ion implanting to Strain survival rate；

Fig. 2 is N of the present invention⁺Ion implanting is mutated to bacterial strain and the influence of positive mutation rate；

Fig. 3 is influence curve of the plasma mutagenesis of the present invention to Strain survival rate；

Fig. 4 is influence of the plasma mutagenesis of the present invention to bacterial strain mutation rate and positive mutation rate；

Fig. 5 is the present invention through low energy N⁺The alkaline egg that ion implantation technique and atmospheric pressure at room plasma induced-mutation technique obtain The shake flask fermentation producing enzyme curve of white enzyme superior strain L-7；

Fig. 6 is the 60T ferment tank producing enzyme curve of alkali protease superior strain L-7 of the present invention；

Fig. 7 is alkali protease electrophoretogram of the present invention；

Wherein, A.Marker, B. basic protein zymoprotein after purification, C. fermentation liquid；

Fig. 8 is opposite enzyme activity of the alkali protease of the present invention at different pH；

Fig. 9 is the opposite enzyme activity of alkali protease of the present invention at different temperatures；

Figure 10 is remnant enzyme activity of the alkali protease of the present invention at different pH after 40 DEG C of heat preservation 1h；

Figure 11 be alkali protease of the present invention at pH11,40,50,60 DEG C respectively heat preservation 2h remnant enzyme activity.

Specific embodiment

Below by specific embodiment, the invention will be further described, and it is not limit that following embodiment, which is descriptive, Qualitatively, this does not limit the scope of protection of the present invention.

Following embodiment used mediums:

(1) preservation/activation medium (g/L): beef extract 8, yeast extract 2, polyprotein peptone 5, NaCl 2, agar 17, Casein 4, K₂HPO₄18, pH is natural.

(2) seed culture medium (g/L): yeast extract 5, tryptone 5, glucose 10, K₂HPO₄18, pH is natural.

(3) fermentation medium (g/L): yeast extract 17, cottonseed meal 30, maltodextrin 100, sodium citrate 3, calcium chloride 2.6, K₂HPO₄18, pH is natural.

(4) milk culturemedium (g/L): skimmed milk power 100, agar 20, pH are natural.

Embodiment 1, induction mutation of bacterium

1, the screening of starting strain

From the Bacillus clausii access seed training that one ring of picking is newly cultivated on inclined-plane (being obtained by this laboratory screening) In nutrient solution (seed culture medium), 12h is cultivated in 34 DEG C, 200r/min, is then applied to solid medium (seed culture medium addition 1% agar) in, continue in 34 DEG C of incubator cultivate 48h, therefrom 20 single colonies of picking are transferred into inclined-plane, then by this 20 A inclined-plane bacterial strain is connected in seed culture medium, one single colonie of every bottle (50mL seed culture medium/250mL triangular flask) inoculation, 200r/min, 34 DEG C of culture 12h, observation bacterium solution become cloudy, after free from extraneous odour by 2% (v/v) inoculum concentration be forwarded to liquid fermentation training Support base.It 34 DEG C, 200r/min, shaken cultivation 50h in baffle flask (50mL fermentation medium/250mL baffle flask), will After fermentation liquid centrifugation, crude enzyme liquid vigor is measured with Forint phenol method, the results are shown in Table 1.

The enzyme activity of 1 original strain of table

For original starting strain after rejuvenation, the enzyme activity average value of strain fermentating liquid is 48000U/mL~52000U/mL. After measuring basic protein enzyme activity, selects enzyme activity higher and more stable bacterial strain is as starting strain.Y-18 highest is 52000U/mL。

The higher bacterial strain Y-18 of enzyme activity in above-mentioned test is subjected to passage test, 20 investigation bacterial strain heredity of continuous passage The stability of shape.As a result (being shown in Table 2): this bacterial strain high yield and stability is good, be suitable as ion implantation mutagenesis goes out bacterium germination Strain.

2 starting strain of table passes on test result

2、N⁺Ion implantation mutagenesis

(1) pre-treatment of sample: by Bacillus clausii starting strain Y-18 in production gemma culture medium (seed culture Base) in culture to produce the gemma phase, take 0.1mL spore solution to be equably coated on aseptic flat board (seed culture medium), use is sterile After wind is dry, plate is then put into low energy accelerator target chamber, target chamber vacuumizes, with energy be 30keV various dose, N⁺ Ion beam is injected.

(2) ion implanting conditions: Implantation Energy 30keV, target chamber vacuum degree are 5-6 × 10^-3Kpa, with 6s pulsed note Enter, be divided into 60s, bolus injection dosage is 1 × 10¹⁴N⁺ions/cm²；Implantation dosage is respectively 5,10,15,20,30,40, 50,70,100,200.Each implantation dosage does a vacuum control.

Each processing and its corresponding vacuum compare forvacuum 5min before ion implantation, to eliminate because being dried in vacuo journey The different possible influences of degree；

(3) N the post-processing of sample: will be passed through⁺The culture dish of culture dish and the vacuum control of ion implanting is used sterile respectively Water elution prepares bacteria suspension, is coated in activation plate (activation medium) after appropriate dilution, sets and counts after cultivating 48h at 34 DEG C Number, for the counting of single colonie and selecting for survival rate.

Survival rate is through N⁺The surviving colonies number of ion implanting processing and the ratio of the surviving colonies number through vacuum control treatment Value.

(4) drafting of Survival curves: the bacteria suspension 1mL after drawing ion implanting, injection are equipped with the test tube of 9mL sterile water In, it is made 10^-1Dilution；Pressure-vaccum 10 repeatedly^-1Dilution, so that it is drawn 1mL after mixing, injection is equipped with 9mL sterile water Test tube in, be made 10^-2Dilution；It is operated by above procedure, preparation 10^-3Dilution；The dilution of suitable concentration is selected, point 0.1mL is not taken to be coated in plate, each dilution does 3 in parallel；After cultivating 48h in 34 DEG C of constant incubators, meter is taken out Number.

Using implantation dosage as abscissa, survival rate is ordinate, draws the relation curve of survival rate and implantation dosage.

Survival rate (%)=N⁺Inject processing clump count/vacuum control clump count × 100%.As a result (see Fig. 1): N⁺Ion implantation dosage reaches 30 × 10¹⁴N⁺ions/cm²Before, Strain survival rate is with N⁺The increase of ion implantation dosage and drop rapidly It is low；Implantation dosage is more than 200 × 10¹⁴N⁺ions/cm²Afterwards, survival rate is substantially zeroed.Work as N⁺Ion implantation dosage is 30 × 10¹⁴ ~50 × 10¹⁴N⁺ions/cm²When in range, " saddle-shape " variation tendency of high-low-high, this spy is presented in the survival rate of strain Some variations are considered as damage effect and protection and stimulation comprehensive function under quality, charge effect under energy, momentum Result.

(5)N⁺Influence of the ion implanting to bacterial strain mutation rate

The ratio between the transparent loop diameter of bacterium colony and the colony diameter under each implantation dosage situation of change is detected using first sieve method, is advised The mutant strain that fixed transparent loop diameter is greater than control strain 5% is known as direct mutation, the mutation that is negative less than 5%, does not dash forward between ± 5% Become.Test result is shown in Fig. 2.

As shown in Figure 2, with the increase of ion implantation dosage, bacterial strain mutation rate increases therewith；Implantation dosage is 30 × 10¹⁴ ~50 × 10¹⁴N⁺ions/cm²The positive mutation rate of bacterial strain is higher (22%~26%) in range, and implantation dosage is more than 50 × 10¹⁴N⁺ ions/cm²After, the mutation rate of bacterial strain continues growing, but its positive mutation rate is in reduced trend.Analyze reason, it may be possible to by Bacterial strain after low dosage injection is easy to happen back mutation, and high dose makes the damage of the large biological molecules such as DNA and biomembrane Effectively cause higher mutation rate and lower positive variation rate.In " saddle " region, injection dosage is suitable for that mutation rate is relatively Height, positive mutation rate are also higher.

3, by N⁺The superior strain of ion implantation mutagenesis screens for the first time

It is coated with the plate of bacterium solution (the i.e. N under best implantation dosage⁺Ion implantation dosage is 40 × 10¹⁴N⁺ions/cm² When) carry out N⁺Ion implantation mutagenesis processing is coated on primary dcreening operation plate after appropriate dilution.

Primary dcreening operation selects double-layer plate, and lower layer is fermentation solid culture medium (fermentation medium adds 1% agar), and upper layer is ox Milk culture medium, by select at random 100 plants of bacterium number consecutively H-1-H-100, one-to-one point is connected to plate lower layer culture medium； Every plate point connects 5 plants of mutagenic bacterias, and every plant of bacterium makees a Zhi Pinghang, and point connects original bacteria and compares, will be quantitative after 34 DEG C of culture 48h Milk culturemedium is uniformly poured on plate, and 10 DEG C of placement 12h calculate the ratio between transparent loop diameter and colony diameter, select ratio Greater than the mutant strain of original bacteria.3 wheel of according to said method screening, tentatively selected six plants of H-3, H-6, H-33, H-66, H-78, H-90 prominent Mutant carries out secondary screening.

4, by N⁺The screening again of the superior strain of ion implantation mutagenesis

6 plants of bacterium that primary dcreening operation is sieved carry out shake flask fermentation culture (fermentation medium) respectively, using 50h enzyme activity as secondary screening Standard, in triplicate.Each mutant strain enzyme activity is as shown in table 3, wherein mutant strain H-6 producing enzyme vigor highest, reaches 65000U/mL, 25% is improved than initial strains.

Table 3 is through N⁺The secondary screening result of the alkali protease superior strain of ion implantation mutagenesis

5, the determination of alkali protease superior strain

To mutant strain H-6 continuous passage 5 times, using 50h fermentation crude enzyme liquid vigor as standard, the results are shown in Table 4, passage five After secondary, enzymatic productivity keeps stablizing, and the merit of bacterial strain can stablize heredity, and mutant strain H-6 is that high yield alkali protein is prominent Become bacterial strain.

Table 4H-6 genetic stability

	Enzyme activity (U/mL)	Opposite enzyme activity (%)
			F1	65000	100
F2	64675±649	99.5±0.8
			F3	64935±456	99.9±0.6
F4	65845±828	101.3±1.0
			F5	66300±729	102.0±0.9

6, plasma mutagenesis

(1) N the pre-treatment of sample: will be passed through⁺The enhanced variant H-6 of ion implantation mutagenesis screening is in production gemma culture Culture disperses after spore solution is made in the fresh bacterium solution of enhanced variant H-6 to the gemma phase is produced in base (seed culture medium) Uniformly, adjusting spore solution concentration with physiological saline is 10⁵-10⁶mL^-1.The spore solution for taking 10uL is added dropwise cold in sterilizing But on the glass slide after.

(2) plasma mutagenic condition: by the slide glass placement region ultraviolet sterilization 30min on objective table, by sample obtained Slide glass is placed on microscope carrier, makes slide glass and jet exit distance 2mm；Open working gas valve, working gas i.e. electric discharge gas Body is helium；External source gas is opened, the radio-frequency voltage of additional 200V, 13.56MHz, jet temperature is room temperature at this time.To sample into Row irradiation, irradiation time 80-180s.

(3) post-processing of sample: the gemma after mutagenesis is flat with activation is coated under sterile washing, after appropriate dilution On plate (activation medium), sets and counted after cultivating 48h at 34 DEG C, for the counting of single colonie and selecting for survival rate.

Survival rate is the ratio of the surviving colonies number after plasma mutagenesis and the surviving colonies number before mutagenesis.

(4) drafting of Survival curves: the spore solution 1mL after drawing plasma mutagenesis, injection is equipped with 9mL sterile water In test tube, it is made 10^-1Dilution；Pressure-vaccum 10 repeatedly^-1Dilution, so that it is drawn 1mL after mixing, injection equipped with 9mL without In the test tube of bacterium water, it is made 10^-2Dilution；It is operated by above procedure, preparation 10^-3Dilution；Select the dilution of suitable concentration Liquid takes 0.1mL to be coated in plate respectively, and each dilution does 3 in parallel；After cultivating 48h in 34 DEG C of constant incubators, take It counts out.

Using mutation time as abscissa, survival rate is ordinate, draws the relation curve of survival rate and mutation time.

Clump count × 100% before clump count/processing after survival rate (%)=plasma mutagenic treatment.As a result (see figure 3): before ARTP irradiation time reaches 120s, Strain survival rate is reduced rapidly with the increase of ARTP irradiation time；Irradiation time After 180s, survival rate is substantially zeroed.When ARTP irradiation time is within the scope of 120~160s, the survival rate of strain is presented " saddle-shape " variation tendency of high-low-high.

(5) influence of the plasma mutagenesis to bacterial strain mutation rate

The ratio between the transparent loop diameter of bacterium colony and colony diameter under ARTP difference irradiation time variation feelings are detected using first sieve method Condition is, it is specified that the mutant strain that transparent loop diameter is greater than control strain 5% is known as direct mutation, the mutation that is negative less than 5%, between ± 5% It is unmutated.Test result is shown in Fig. 4.

As shown in Figure 4, with the increase of ARTP irradiation time, bacterial strain mutation rate increases therewith；Irradiation time is in 120- The positive mutation rate of bacterial strain is higher (18%~26%) within the scope of 160s, and after irradiation time 140s, the mutation rate of bacterial strain continues to increase Add, but its positive mutation rate is in reduced trend.Analyze reason, it may be possible to since the bacterial strain after irradiation time is too long is easy to happen back Multiple mutation, and prolonged plasma irradiation makes the damage of the large biological molecules such as DNA and biomembrane effectively cause higher mutation Rate and lower positive variation rate.In " saddle " region, ARTP irradiation time be suitable for, mutation rate is relatively high, positive mutation rate also compared with It is high.Finally determine that optimal ARTP irradiation time is 140s.

7, high productive mutant H-6 using the double mutations superior strain after plasma mutagenesis first screening

By the bacteria suspension of high productive mutant H-6 in best ARTP irradiation time (when i.e. ARTP irradiation time is 140s) mutagenesis Processing is coated on primary dcreening operation plate (activation medium) after appropriate dilution.

Primary dcreening operation selects double-layer plate, and lower layer is fermentation solid culture medium (fermentation medium), and upper layer is milk culturemedium, will The 100 plants of bacterium number consecutively L-1-L-100 selected at random, one-to-one point are connected to plate lower layer culture medium；Every plate point connects 5 plants Mutagenic bacteria, every plant of bacterium makees a Zhi Pinghang, and point connects original bacteria and compares, after 34 DEG C of culture 48h, quantitative milk culturemedium is equal Even to be poured on plate, 10 DEG C of placement 12h calculate the ratio between transparent loop diameter and colony diameter, and every plate selects 2 ratios and is greater than original The mutant strain of beginning bacterium.3 wheel of according to said method screening, tentatively selected six plant mutant strain of L-2, L-7, L-32, L-65, L-77, L-89 into Row secondary screening.

8, the screening again of double mutations superior strain

6 plants of bacterium that primary dcreening operation is sieved carry out shake flask fermentation culture (fermentation medium) respectively, using 50h enzyme activity as secondary screening Standard, in triplicate.Each mutant strain enzyme activity is as shown in table 5, wherein mutant strain L-7 producing enzyme vigor highest, reaches 79000U/mL, 51.9% is improved than initial strains.

The secondary screening result of alkali protease superior strain of the table 5 after duplex mutagenesis

9, the determination of alkali protease superior strain

Continuous passage test is carried out to the high yield double mutant screened in the above mutagenesis testing, surveys its enzyme activity, The stability for investigating its inhereditary feature screens the bacterial strain that can be finally applied in production.

To mutant strain L-7 continuous passage 5 times, using 50h fermentation crude enzyme liquid vigor as standard, the results are shown in Table 6, passage five After secondary, enzymatic productivity keep stablize, this with report after ion implantation mutagenesis and plasma mutagenesis, the merit of bacterial strain Can stablize heredity it is consistent, mutant strain L-7 is high yield alkali protein bacterial strain.

Table 6L-7 genetic stability

	Enzyme activity (U/mL)	Opposite enzyme activity (%)
			F1	79000	100
F2	78642±649	99.5±0.8
			F3	78914±456	99.9±0.6
F4	80006±828	101.3±1.0
			F5	80552±729	102.0±0.9

Embodiment 2, the fermentation of alkali protease superior strain shaking flask level produce protease

(the 50mL seed training from the Bacillus clausii L-7 access seed culture medium that one ring of picking is newly cultivated on inclined-plane Support base/250mL triangular flask), 200r/min, 34 DEG C of culture 12h, 2% (v/v) inoculum concentration are forwarded to fermentation medium, and 34 DEG C, 200r/min, shaken cultivation in baffle flask (50mL fermentation medium/250mL baffle flask), at interval of certain time Afterwards, fermentation liquid is centrifuged, crude enzyme liquid vigor is measured with Forint phenol method, as a result shown in attached drawing 5.

Embodiment 3, alkali protease superior strain 60T ferment tank technique

(the 100mL seed training from the Bacillus clausii L-7 access seed culture fluid that one ring of picking is newly cultivated on inclined-plane Support base/500mL baffle flask), 200r/min, 34 DEG C of culture 12h transfer according to the inoculum concentration of 5% (v/v) and ferment into 60T Tank, fermentation tank capacity 70% (fermentation medium).Temperature: 34 DEG C；Revolving speed: 300r/min；Ventilation quantity: 1: 1.5；Dissolved oxygen maintains 25%.The hydrochloric acid of auto-feeding ammonium hydroxide and 20% (v/v) in fermentation process, make fermentation liquid pH value maintain 7.0.Using the work Skill is continuously fermented three batches, and as shown in Fig. 6, discovery is in the case where the feeding method controls technique, and after 30h, alkali protease is a large amount of Synthesis, when reaching 55h, enzyme activity highest 88000U/mL.After 54h, producing enzyme slows, and vigor decline, thallus enters quickly The extinction phase, while pH value rises rapidly.

As shown in Fig. 7, SDS-PAGE protein electrophoresis shows sample after purification oneself reaches that electrophoresis is pure, and molecular weight is 28kDa。

Embodiment 4 studies the property of enzyme

(1) enzyme activity determination

1. the measurement of basic protein enzyme activity

With light industry ministry standard QB/T1803-93 (Folin reagent color developing method): 1 milliliter of enzyme solution is at 40 DEG C, pH11, often It is 1 enzyme activity unit (U/mL) that minute reaction, which generates enzyme amount required for 1 μ g tyrosine,.

2. the drafting of standard curve

Prepare the tyrosine standard solution (0,10,20,30,40,50,60 μ g/mL) of various concentration.Take tyrosine standard molten Liquid 1.00mL adds sodium carbonate liquor 5.00ml, the Folin reagent solution 1.00mL of 0.4mol/L, is placed in 40 ± 0.2 DEG C of water-baths Develop the color 20min.It takes out, measures its absorbance A respectively with spectrophotometer₆₈₀(using the pipe without tyrosine as blank).With extinction Spend A₆₈₀For ordinate, the concentration c of tyrosine is abscissa, is drawn standard curve (this line should pass through zero point).

3. determination step

(1) fermentation liquid 8000r/min is centrifuged 10min, supernatant is taken to be diluted to borax-sodium hydroxide buffer solution Debita spissitudo, as enzyme solution to be measured.

(2) casein, solution of trichloroacetic acid are put into 40 ± 0.2 DEG C of waters bath with thermostatic control, preheat 5min.

(3) following procedure operation is pressed:

4. enzyme activity calculation method

X=A × K × 4/10 × n=0.4 × A × K × n

X-sample enzyme activity, u/mL

A-sample parallel test mean light absorbency

K-extinction constant

The total volume of 4-reaction reagents, mL

N-extension rate

(2) purifying process of enzyme: fermentation liquid 100ml, 8000r/min is taken to be centrifuged 10min.Supernatant is taken, with saturation degree By saltouing, 8000r/min is centrifuged 10min and collects precipitating 70% solid sulphuric acid.Precipitating is dissolved in Tris-HCI (pH8.8) In buffer, dialysis desalination is carried out in identical buffer.It is centrifuged off a small amount of insoluble matter, supernatant is taken to cross DEAE- SepharoseCL-6B ion exchange column is purified, and is washed with 20mMTris-Hcl (pH8.8 contains 10mMNacl) buffer It is de-.Alkali protease is unadsorbed, is first eluted, and obtains colorless and transparent liquid.It is dry to collect enzyme solution progress vacuum refrigeration It is dry, it obtains pure enzyme powder and is further analyzed.

(3) zymologic property research

Using casein as substrate, with light industry ministry standard QB/T1803-93 (Folin reagent color developing method), i.e. forint (Folin) method；Optimal pH, optimum temperature, pH stability and the temperature stability of the alkali protease are studied respectively.

It measures optimal pH: under the conditions of 40 degree, measuring alkali protease respectively under pH5,6,7,8,9,10,11,12,13 Opposite enzyme activity (attached drawing 8).

It measures optimum temperature: under conditions of pH11, measuring alkali protease respectively 30,40,50,60,70,80,90 Opposite enzyme activity (attached drawing 9) at DEG C.

Measure pH stability: after enzyme solution is kept the temperature 1h under the conditions of pH5,6,7,8,9,10,11,12,13 and 40 DEG C, most Remnant enzyme activity (attached drawing 10) is measured under the conditions of thermophilic degree and optimal pH；Remnant enzyme activity refers to be kept the temperature under the conditions of different pH and 40 DEG C The enzyme activity value that enzyme solution afterwards measures under optimum condition, the enzyme activity value measured under optimum condition relative to enzyme solution when not keeping the temperature Percentage.

Measuring temperature stability: after enzyme solution is kept the temperature 2h under conditions of 40,50,60 DEG C and pH11, in optimum temperature and Remnant enzyme activity (attached drawing 11) is measured under the conditions of optimal pH；Remnant enzyme activity refers to the enzyme after keeping the temperature under the conditions of different temperatures and pH11 The enzyme activity value that liquid measures under optimum condition, the percentage relative to the enzyme activity value that enzyme solution measures under optimum condition when not keeping the temperature Number.

Conclusion: as shown in Figure 8: the most suitable action pH of the enzyme is 11；

As shown in Figure 9: the optimum temperature of the enzyme is 40 DEG C；

As shown in Figure 10: the enzyme is stablized in the range of pH6~12；

As shown in Figure 11: the enzyme has preferable thermal stability at 40~50 DEG C or so.

Claims

1. a kind of alkali protease superior strain, it is characterised in that: the bacterial strain is specially gram Lloyd's bacillus (Bacillus Clausii) L-7, deposit number are as follows: CGMCC No.12953.

2. application of the alkali protease superior strain described in claim 1 in production alkali protease.

3. the application of alkali protease superior strain described in claim 2, which is characterized in that the strain fermentation produces alkaline egg The method of white enzyme is as follows:

(1) seed culture: from gram Lloyd's bacillus L-7 access seed culture fluid that one ring of picking on inclined-plane is newly cultivated, 200r/min, 34 DEG C of culture 12h；

(2) fermented and cultured: transferring according to 5% inoculum concentration into fermentor, and fermentation tank capacity is 70% fermentation medium, temperature Degree: 34 DEG C；Revolving speed: 100~600r/min；Ventilation quantity: 1: 0.5~1: 2.5；Dissolved oxygen maintains 20~30%；In fermentation process The hydrochloric acid of auto-feeding ammonium hydroxide and 20% (v/v), makes fermentation liquid pH value maintain 7.0；Fermentation time 50-60h；

The seed culture medium are as follows: yeast extract 5g/L, tryptone 5g/L, glucose 10g/L, K₂HPO₄18g/L, pH are natural；

The fermentation medium are as follows: yeast extract 17g/L, cottonseed meal 30g/L, maltodextrin 100g/L, sodium citrate 3g/L, Calcium chloride 2.6g/L, K₂HPO₄18g/L, pH are natural.