CN107129978A - The fermentation process of heat-resisting lysine aminopeptidase - Google Patents
The fermentation process of heat-resisting lysine aminopeptidase Download PDFInfo
- Publication number
- CN107129978A CN107129978A CN201710237381.9A CN201710237381A CN107129978A CN 107129978 A CN107129978 A CN 107129978A CN 201710237381 A CN201710237381 A CN 201710237381A CN 107129978 A CN107129978 A CN 107129978A
- Authority
- CN
- China
- Prior art keywords
- resisting
- heat
- fermentation process
- lysine
- lysine aminopeptidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a kind of fermentation process of heat-resisting lysine aminopeptidase：Recombinant yeast pichia pastoris seed liquor is seeded to fermented and cultured in BSM culture mediums by 10% inoculum concentration；After the initial glycerine in culture medium has consumed, glycerol adding supplemented medium is flowed, works as OD600Stop stream after reaching 400 to add, then stream plus methanol aqueous solution carry out Fiber differentiation, stop fermented and cultured when enzyme activity declines, obtain heat-resisting lysine aminopeptidase.The recombinant yeast pichia pastoris bacterium of the present invention is the Pichia yeast containing the lysine aminopeptidase gene from pseudomonas aeruginosa NJ 814, and pseudomonas aeruginosa NJ 814 deposit number is CGMCC No.7638.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of fermentation process of heat-resisting lysine aminopeptidase.
Background technology
Lysine aminopeptidase has very heavy as a kind of circumscribed-type protease in multiple fields such as food, medicine and chemical industry
The application wanted.Available for the bitter taste removed in protein hydrolyzate, the depth hydrolysis of protein, the preparation of biologically active peptide or it is used as
Recombinant protein fixative or protein sequence determine required toolenzyme etc., in food, medicines and health protection and chemical industry application prospect
It is wide.
Pichia pastoris, as a kind of host being widely used, successfully expresses hundreds of foreign protein, can
High density fermentation is carried out, the expression of foreign protein is improved.Compared with traditional shake flask fermentation, Pichia pastoris high density fermentation
With following features：(1) higher biomass；(2) space needed for reaction system is small, and cost is low；(3) can by provide pure oxygen and
Feed supplement, makes dissolved oxygen and nutrition in fermentation process more fully meet thalline producing enzyme.
The enzyme of proteolysis for possessing heat-resisting feature has apparent advantage in terms of extraction and protein hydrolysate raw material.For example
Cooling is not required to after being pre-processed before raw material hydrolysis just can be more beneficial for the fast of proteolytic enzyme with enzyme-added hydrolysis after protein structure denaturation
Speed effect, substantially increases hydrolysis efficiency, and lysine is used as the conventional ammonia in a kind of necessary amino acid and enriched food
Base acid, the Research Significance in terms of its preparation and application is great.But at present using the Pichia pastoris fermentation heat-resisting lysine ammonia peptide of output
The procedure of enzyme is more complicated, and yield is not high, significantly limit its application and studies.
In addition, when separating heat-resisting lysine aminopeptidase using prior art, it may appear that the phenomenon for inactivating enzyme, thus also limit
The application study of the enzyme is made.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of fermentation process of heat-resisting lysine aminopeptidase,
Using the fermentation process of the present invention, it is possible to increase the expression of extracellular lysine aminopeptidase, and the purification step energy of the present invention
When enough ensureing the active unaffected of lysine aminopeptidase, bad sour aminopeptidase of the invention and restriction endonuclease synergetic hydrolysis albumen, energy
It is enough first to hydrolyze lysine, leucine is then hydrolyzed again.
On the one hand, the invention provides a kind of fermentation process of heat-resisting lysine aminopeptidase, comprise the following steps：
(1) recombinant yeast pichia pastoris seed liquor is seeded to fermented and cultured in BSM culture mediums by 8-12% inoculum concentrations, restructuring is finished
Red yeast is the Pichia yeast containing the lysine aminopeptidase gene from pseudomonas aeruginosa NJ-814, P. aeruginosa
Bacterium NJ-814 deposit number is CGMCC No.7638；
(2) after the initial glycerine in culture medium has consumed, glycerol adding supplemented medium is flowed, works as OD600Reach after 350-450
Stop stream adding, then stream plus methanol aqueous solution carry out Fiber differentiation, stop fermented and cultured when enzyme activity declines, obtain heat-resisting bad ammonia
Sour aminopeptidase.
Further, it is 450-550rmin in initial speed in step (1)-1, throughput is 2vvm, 30 DEG C of bar
Fermented and cultured under part.
Further, in step (2), the flow acceleration of glycerine supplemented medium is 17-19mLh-1·L-1。
Further, in step (1), the preparation method of recombinant yeast pichia pastoris seed liquor comprises the following steps：Will activation
Recombinant yeast pichia pastoris afterwards is in BMGY culture mediums, in 25-35 DEG C, 200-250rmin-1Rotating speed under cultivate.
Further, in step (2), the step of also including hungry culture 1.5-2.5h before stream plus methanol aqueous solution,
Do not flowed plus any carbon source during starvation culture.
Further, in step (2), when stream plus methanol aqueous solution, in 1-2h, methanol aqueous solution flow acceleration is
1.0-1.5mL·h-1·L-1, during 2h-3h, methanol aqueous solution flow acceleration 1.5-2.5mLh-1·L-1, constant speed stream adds after 3h,
And constant speed flow acceleration is 2.5-3.0mLh-1·L-1.Add control in the low flow velocity stream at induction initial stage, it is to avoid too high first
Toxic action of the determining alcohol to recombination yeast, is conducive to methanol using the synthesis of enzyme system and steps up tolerance of the thalline to methanol
Ability.
Further, in step (2), glycerine supplemented medium includes the glycerine and 1.0- that mass fraction is 45-55%
1.5% PTM1.It is preferred that, the mass fraction of glycerine is 50%.
Further, in step (2), the PTM1 that mass fraction is 1.0-1.5% is contained in methanol aqueous solution.Often
Contain each component of following mass percent in 100mL PTM1 Trace salts solutions：0.008%NaI, 0.3%MnSO4·H2O,
0.6%CuSO4·5H2O, 0.02%Na2MoO4, 0.002% boric acid, 2%ZnSO4·7H2O, 0.05%CoCl2, 6.5%
FeSO4·7H2O, 5mL sulfuric acid, 0.02% biotin.
Further, in step (2), dissolved oxygen is 20%-30% during fermented and cultured.By controlling rotating speed, ventilation
Amount and tank pressure maintain dissolved oxygen.
Further, the gene order of heat-resisting lysine aminopeptidase is as shown in SEQ ID NO.1.
Further, after step (2), in addition to following purification step：
(S1) ammonium sulfate for being 40%-60% with mass fraction is saltoutd heat-resisting lysine aminopeptidase, is then dialysed；
(S2) anion-exchange chromatography is carried out to the product that dialysis is obtained；
(S3) the heat-resisting lysine aminopeptidase liquid that concentration, filtration chromatography are obtained, then through gel permeation chromatography.
Further, in step (S2), anion-exchange chromatography is carried out using Hitrap Q HP anion-exchange columns.
Further, in step (S2), 50mM Tris-HCl buffer solutions are used during anion-exchange chromatography, wherein, remove
When miscellaneous, the NaCl that mass fraction is 28% is contained in buffer solution；When eluting destination protein, it is containing mass fraction in buffer solution
42% NaCl.
Further, in step (S3), the molecular cut off of the film used during filtering is 20-40kDa.
Further, in step (S3), Gel filtration is carried out using the 10/300GL Filter columns of Superdex TM 75
Analysis.
Further, in step (S1), the crude enzyme liquid that heat-resisting lysine aminopeptidase can also obtain for shake flask fermentation.
The preparation method of crude enzyme liquid comprises the following steps：By the single bacterium colony of recombinant yeast pichia pastoris in BMGY culture mediums
18h is cultivated under 230rpm, is then transferred in BMMY culture mediums and cultivates 120h under conditions of 25 DEG C, pH=5.0, every 24h to
The methanol that final concentration 1.5% is added in nutrient solution is induced, and after obtained zymotic fluid is centrifuged, supernatant is lysine ammonia
Peptase crude enzyme liquid, wherein recombinant yeast pichia pastoris are to contain the lysine aminopeptidase gene from pseudomonas aeruginosa NJ-814
Pichia yeast, pseudomonas aeruginosa NJ-814 deposit number is CGMCC No.7638, and Pichia yeast is finished red for Pasteur
Yeast strain GS115.
Due to N-terminal and the C-terminal of heat-resisting lysine aminopeptidase of the present invention hang it is histidine-tagged after, its activity can be by
Influence, therefore purified using the above method.
Using the heat-resisting lysine aminopeptidase hydrolyzable casein prepared by above-mentioned purification process, following step is specifically included
Suddenly：
By heat-resisting lysine aminopeptidase and alkali protease synergetic hydrolysis casein at 80 DEG C, hydrolysis time is 3h, junket
The concentration of albumen is 10%, and the addition of heat-resisting lysine aminopeptidase is 90U/g.
Further, every liter of BMGY culture medium includes each component of following quality：Peptone 20g, glycerine 10g, yeast
Cream 10g, YNB (no amino yeast nitrogen)) 13.4g, biotin 0.4g, 100mM phosphate buffer (pH 6.0).
Further, every liter of BMMY culture medium includes each component of following quality：Peptone 20g, methanol 10g, yeast
Cream 10g, YNB 13.4g, biotin 0.4g, 100mM phosphate buffer (pH 6.0).
Further, every liter of BSM culture medium includes each component of following mass percent：4% glycerine, 1.49%
MgSO4·7H2O, 0.413%KOH, 1.82%K2SO4, 2.67%85% phosphoric acid, 0.093%CaSO4, 0.435%PTM1.
Further, each component of following mass percent is contained in every 100mL PTM1 Trace salts solutions：0.008%
NaI, 0.3%MnSO4·H2O, 0.6%CuSO4·5H2O, 0.02%Na2MoO4, 0.002% boric acid, 2%ZnSO4·7H2O,
0.05%CoCl2, 6.5%FeSO4·7H2O, 5mL sulfuric acid, 0.02% biotin.
Recombinant yeast pichia pastoris of the present invention is as the side disclosed in the Chinese patent of Application No. 201510379603.1
Constructed by method.Pseudomonas aeruginosa NJ-814 deposit number is that CGMCC No.7638 are preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number is CGMCC No.7638.
By such scheme, the present invention at least has advantages below：
The invention provides a kind of fermentation process in high density of the fermentation process of heat-resisting lysine aminopeptidase, effectively solving
The heat-resisting lysine aminopeptidase gene can not further improve lysine aminopeptidase on the basis of the problem of high efficient expression in bacterium
Yield；It is affected because the enzyme upstream builds N-terminal with the histidine-tagged rear activity of C-terminal extension, therefore metal can not be used during purifying
Affinity chromatography is chelated, the present invention is using the combination purification schemes under the specified conditions through optimizing acquisition repeatedly：Salt fractionation-
The Hitrap Q HP ion-exchange chromatographies-gel permeation chromatographies of Superdex 75, the enzyme purified solves above-mentioned problem,
So as to which characteristic, structure effect to study heat-resisting lysine aminopeptidase etc. provides good basis；The invention provides heat-resisting bad ammonia
Application of the sour aminopeptidase in caseinhydrolysate raw material, is that the research for further widening the enzyme synergetic hydrolysis protein raw materials is established
Good basis.
The lysine aminopeptidase of present invention fermentation output, not only with good genetic stability, and with good temperature
Stability is spent, is that good basis has been established in the industrialization of aminopeptidase and the application in field of food.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the growth curve of glycerol feed phase recombinant yeast pichia pastoris；
Fig. 2 is relation curve of the yield with the methanol induction time of lysine aminopeptidase；
Fig. 3 is the electrophoretogram of the enzyme of different degree of purification；
Fig. 4 is the SDS-PAGE test results of lysine aminopeptidase after purification；
Fig. 5 is the enzyme activity test result of lysine aminopeptidase at different temperatures after purification；
Fig. 6 illustrates the temperature stability result of lysine aminopeptidase after purification.
Embodiment
With reference to the accompanying drawings and examples, the embodiment to the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
(1) the enzyme activity determination method of lysine aminopeptidase of the present invention is as follows：
Using L-Leu-paranitroanilinum as substrate, 2mL pH=9.0 Tris-HCl buffer solutions are added, take 1mL to dilute
Enzyme liquid and 1mL substrates (concentration of substrate is 2mM), 10min is reacted in 80 DEG C, and light absorption value is then determined at 405nm, calculates enzyme activity
Power.Wherein, the enzyme activity of lysine aminopeptidase refers to：Under certain condition, its decomposition 1B per minute-paranitroanilinum production
Enzyme amount required for raw 1 μM paranitroanilinum, as one enzyme-activity unit, calculation formula is as follows：
Wherein Abs is light absorption value at 405nm.
(2) thalline weight in wet base assay method of the present invention：Accurate 1mL bacterium solutions of drawing in the 2mL EP pipes weighed, at a high speed from
Supernatant discarding after heart 5min, it is that (unit is gmL to yeast weight in wet base that gained weight, which subtracts sky centrifuge tube weight,-1)。
(3) culture medium used in the present invention is related to following several：
MD culture mediums (g/L.h):Glucose 20, agar 20, YNB 13.4, biotin 0.4；
MM culture mediums (g/L):Methanol 5mL/L, agar 20, YNB 13.4, biotin 0.4；
BMGY culture mediums (g/L):Peptone 20, glycerine 10, yeast extract 10, YNB 13.4, biotin 0.4,100mM phosphoric acid
Salt buffer pH 6.0；
BMMY culture mediums (g/L):Peptone 20, methanol 10, yeast extract 10, YNB 13.4, biotin 0.4,100mM phosphoric acid
Salt buffer pH 6.0；
The upper tank culture mediums of BSM：4% glycerine, 1.49%MgSO4·7H2O, 0.413%KOH, 1.82%K2SO4, 2.67%
85% phosphoric acid, 0.093%CaSO4, 0.435%PTM1.
Glycerine supplemented medium：50% glycerine, 1.2%PTM1.
PTM1 Trace salts solutions (100mL)：0.008%NaI, 0.3%MnSO4·H2O, 0.6%CuSO4·5H2O,
0.02%Na2MoO4, 0.002% boric acid, 2%ZnSO4·7H2O, 0.05%CoCl2, 6.5%FeSO4·7H2O, 5mL sulfuric acid,
0.02% biotin, filtering with microporous membrane, 4 DEG C of preservations.
The bacterial strain that the present invention is used includes：E. coli JM109, Pichia pastoris GS115, Application No.
201510379603.1 Chinese patent disclosed in method build recombinant yeast pichia pastoris bacterium.
The high density fermentation of the heat-resisting lysine aminopeptidase of embodiment 1
Using recombinant yeast pichia pastoris bacterium as fermentation strain, specific fermentation process is as follows：
(1) recombinant yeast pichia pastoris of picking activation (co-cultures 18 bottles) into 25mL BMGY shaking flasks, in 30 DEG C, 230r
min-1Shaking table in cultivate 20h, gained zymotic fluid is upper tank seed liquor.
(2) the 7L fermentation tank sterilization treatments of 4L BSM culture mediums are will be equipped with, after after instrument connection, pump Feeding ammonia water is electric to pH
The show value of pole is 5.0, and it is 500rmin to control initial speed-1, throughput is 2vvm, and fermentation temperature is 30 DEG C.It is added portionwise
17.4mL crosses the degerming PTM1 solution of film and seed liquor, and seed liquor is seeded in fermentation tank by 10% inoculum concentration, calibration dissolved oxygen electricity
Pole.By controlling rotating speed, throughput and tank pressure to maintain dissolved oxygen more than 20%.
(3) DO values are the important parameters for reflecting yeast growth, and DO values start very fast rise and show glycerine in zymotic fluid
Consumption is complete, now needs to add glycerine to obtain more thalline.This stage stream adds containing 12mLL-1PTM1 glycerine feed supplement
The mass fraction of culture medium, wherein glycerine is 50%, and glycerine supplemented medium flow acceleration is 18.15mLh-1·L-1, periodically
Each parameter is surveyed in sampling.
(4) OD is worked as600After reaching 400, stop glycerine feed supplement immediately, then hungry culture 2h.Yeast is only in the training without glycerine
Metabolizing methanol in base is supported, so when dissolved oxygen rapidly rises, needing hungry culture a period of time to exhaust glycerine.
(5) after glycerol depletion, plus methanol aqueous solution carries out Fiber differentiation.When starting methanol induction, regulation temperature is 28
DEG C, added with pump fed-batch mode and contain 12mLL-1PTM1 methanol solution.The methanol feeding speed in initial 2h is set to be
1mL·h-1·L-1, methanol feeding speed in methanol feeding speed, 3h is then gradually stepped up by air pump and reaches 3mLh-1·
L-1, hereafter continue to flow plus methanol with the flow acceleration.Methanol induction phase is sampled every 12h, surveys zymotic fluid OD600, thalline it is wet
Weight, aminopeptidase enzyme activity and protein content, when enzyme activity declines, (84h after stream plus methanol) stops fermented and cultured.
Fig. 1 is the growth curve of glycerol feed phase recombinant yeast pichia pastoris, as shown in figure 1, during glycerine feed supplement, hair
Zymotic fluid OD600, thalline weight in wet base and protein content keep rising.Fig. 2 is yield and the methanol induction time of lysine aminopeptidase
Relation curve, Fig. 2 shows, as initial induction bacterium scale of construction OD600For 400 when, induction 84h after supernatant aminopeptidase enzyme activity reach
Highest, it is 7.8UmL-1, it is 2.14 times of shaking flask producing enzyme level.
The heat-resisting lysine aminopeptidase of above-mentioned acquisition is purified, after selection 40%-60% ammonium sulfate is saltoutd
Dialyzed overnight.Then dialyzate is collected, dialyzate is passed through Hitrap Q HP anion-exchange chromatographies, with containing NaCl's
((pH8.5) is eluted 50mM Tris-HCl buffer solutions, first by the buffer solution for containing the NaCl that mass fraction is 28%
Removal of impurities is carried out, then destination protein is eluted with containing mass fraction for 42% NaCl buffer solutions；Collect the elution of enzyme activity highest
Peak, then using the concentration of 30kDa super filter tubes, one is entered after crossing film using the 10/300GL gel permeation chromatographies of Superdex TM 75
Step is isolated and purified, and obtains heat-resisting lysine aminopeptidase after purification.
The purifying and property research of the heat-resisting lysine aminopeptidase of embodiment 2
Recombinant yeast pichia pastoris bacterium is built using the method disclosed in the Chinese patent of Application No. 201510379603.1, will
Recombinant yeast pichia pastoris bacterium single bacterium colony 230rpm in BMGY culture mediums shakes 18h, and accumulation thalline is after 25mL BMMY (pH=
5.0) 25 DEG C of culture 120h in culture medium, methanol induction is carried out every the 24h methanol that final concentration 1.5% is added into nutrient solution.
The zymotic fluid of acquisition is centrifuged into 5min under the conditions of 10000rpm, supernatant is heat-resisting lysine aminopeptidase crude enzyme liquid.
Above-mentioned crude enzyme liquid is subjected to grade ammonium sulfate salting-out with 10% gradient, and detects often step residual enzyme activity, final choosing
Select 40%-60% ammonium sulfate saltoutd after dialyzed overnight.Then dialyzate is collected, dialyzate is passed through Hitrap Q HP
Anion-exchange chromatography, with the Tris-HCl buffer solutions of the 50mM containing NaCl ((pH8.5) is eluted, first by containing
Mass fraction carries out removal of impurities for 28% NaCl buffer solution, is then eluted with containing mass fraction for 42% NaCl buffer solutions
Destination protein；Enzyme activity highest eluting peak is collected, then using the concentration of 30kDa super filter tubes, crosses and Superdex TM is used after film
7510/300GL gel permeation chromatographies are further isolated and purified, and obtain heat-resisting lysine aminopeptidase after purification.Its specific enzyme activity reaches
36.42U/mg, purification is up to 3.95, and purification result is shown in Fig. 3.Fig. 3 is the electrophoretogram of the enzyme of different degree of purification, wherein, figure
Middle M represents Marker (mark)；Electrophoresis result in figure after 1 correspondence gel permeation chromatography；2 corresponding Hitrap Q HP are cloudy in figure
Electrophoresis result after ion-exchange chromatography；In figure 4 the electrophoresis result after ammonium sulfate precipitation is represented in the 3 original zymotic fluid of correspondence, figure.
As seen from the figure, using combination separation means, its degree of purification is greatly improved.
Table 1 is the purification result of heat-resisting lysine aminopeptidase, and table 1 shows, through oversalting, Hitap Q HP column chromatographies and
After purification, specific enzyme activity brings up to 36.41 to Superdex75 column chromatographies from the 9.21 of crude enzyme liquid, and purification is up to 3.95 times.Wherein
Saltout and Q posts eliminate a large amount of foreign proteins, and gel column Superdex 75 has played Refinement to lysine aminopeptidase.
The purification result of the heat-resisting lysine aminopeptidase of table 1
By above-mentioned lysine aminopeptidase after purification and deglycosylating enzyme Endo HfMixing, 37 DEG C of heated at constant temperature 4h.Heating
After denaturation, sampling carries out SDS-PAGE tests, as a result as shown in figure 4, big protein band disappears as can be seen from Figure 4, only deposits
In small protein band, illustrate that the enzyme there occurs partial glycosylation.
Above-mentioned lysine aminopeptidase after purification is placed under different temperatures and carries out enzyme activity determination, and by enzyme liquid in not equality of temperature
1h, 2h and 3h are incubated under degree (30 DEG C, 60 DEG C, 70 DEG C, 80 DEG C) respectively, detection residual enzyme activity defines the highest enzyme activity measured
For 100%, the percentage that other enzyme activity account for highest enzyme activity is relative enzyme activity, as a result as Fig. 5-6, Fig. 5 shows, at 80 DEG C, is relied
The enzyme activity highest of propylhomoserin aminopeptidase, with the rise of temperature, its enzyme activity gradually increases, and temperature is higher than after 80 DEG C, and its enzyme activity slightly has
Decline, it is 80 DEG C to illustrate its optimum temperature.As can be seen from Figure 6, the aminopeptidase is when retaining 2h for 50 DEG C -70 DEG C, and residual enzyme activity exists
More than 80%, illustrate that the restructuring aminopeptidase still has good stability below 70 DEG C.Compared with original bacteria producing enzyme, use
The lysine aminopeptidase is verified in deglycosylation enzyme hydrolysis and electrophoretic analysis, but occur the glycosylation of part, though do not carry further
Its high temperature stability, but have certain help to the high efficient expression of its correct folding and exocytosis.
The enzymolysis application of the heat-resisting lysine aminopeptidase of embodiment 3
10% casein solution is prepared with 50mM pH=9.0 Tris-HCl solution, 3000Ug is added-1(E/S)
Basic protein enzyme powder, 50 DEG C of reactions 3h, boiling water bath 15min, cooling.Add 90Ug-1Heat-resisting lysine aminopeptidase, 80 DEG C
Isothermal vibration 3h, boiling water bath 10min, 12000rmin-1Under the conditions of centrifuging and taking supernatant, then survey hydrolysate polypeptide distribution.
By supernatant and 10% trichloroacetic acid 1:1 mixing, stands 1h, 15000rmin-1Supernatant is taken after centrifugation, amino acid content is surveyed.Knot
Fruit is shown in Table 2-3.
Table 2 is the polypeptide distribution tests result of hydrolysate, as can be seen from the table, and double-enzyme hydrolysis liquid is (i.e. simultaneously using resistance to
Hot lysine aminopeptidase and alkali protease) in below 180Da content of peptides substantially increase, further relatively more double enzymes collaborations with
And two groups of experimental results of inscribe alkali protease are used alone, it is possible to find and another hydrolysis feature of the enzyme, i.e., in having
In the presence of enzyme cutting, enzyme meeting selective hydrolysis lysine hydrolyzes leucine again, uses heat-resisting lysine aminopeptidase and alkali protease
Synergetic hydrolysis compared with the result individually hydrolyzed using restriction endonuclease, the former leucine increase rate is 2.5 times, and lysine
Content then improves 8 times, the rule and feature of this synergetic hydrolysis to the selection of the hydrolysis of specific protein raw material from now on provide according to
According to.
The polypeptide distribution tests result of the hydrolysate of table 2
Table 3 is the amino acid content test result of hydrolysate, as a result shows that total amino acid content is significantly in hydrolyzate
Improve and lysine therein and leucine content improve ratio maximum, meet the hydrolysis characteristic of the enzyme.With not enzyme-added blank
Control (not digesting) is compared, and is significantly increased using the free amino acid total amount in aminopeptidase, alkali protease, double-enzyme hydrolysis liquid
Plus, respectively the 10.5 of blank control times, 22.5 times and 65 times.
The amino acid content test result of the hydrolysate of table 3
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Sequence table
<110>Southern Yangtze University
<120>The fermentation process of heat-resisting lysine aminopeptidase
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1500
<212> DNA
<213>Lysine aminopeptidase gene order
<400> 1
atgggcaaac ccaacccgtc gatctgcaaa tcgccgttgc tggtcagcac cccgcttggc 60
ctgccgcgct gcctgcaagc cagcaacgtg gtcaagcgcc tgcagaagct ggaggacatc 120
gccagtctca acgacggcaa ccgcgccgcc gccacgccgg gctaccaggc ctccgtcgac 180
tacgtgaagc agaccctgca gaaagccggc tacaaggtca gcgtgcagcc cttcccgttc 240
accgcctact acccgaaagg cccgggtagc ctgagcgcca ccgtgccgca gccggtcacc 300
tacgaatggg agaaggactt cacctacctg tcgcagaccg aggcaggcga cgtcaccgcc 360
aaggtggtcc cggtggacct gtccctcggc gccggcaaca cctccaccag cggttgcgag 420
gcggaagact tcgccaactt cccggccggc tcgatcgcgc tgatccagcg cggcacctgc 480
aacttcgagc agaaggccga gaacgccgcg gccgccggcg ccgccggggt gatcatcttc 540
aaccagggca acaccgacga ccgcaagggc ctggagaacg tcaccgtggg cgagtcctac 600
gagggcggca tcccggtgat cttcgccacc tacgacaacg gcgtggcctg gtcgcagacc 660
ccggacctgc agttgcacct ggtggtcgac gtggtacgca agaagaccga gacctacaac 720
gtggtcgccg agacccgtcg cggcaacccg aacaacgtgg tgatggtcgg cgcgcacctc 780
gactcggtgt tcgaaggccc cggtatcaac gacaacggtt cgggcagcgc cgcccaactg 840
gagatggccg tgctgctggc caaggcgctg ccggtcaaca aggtgcgctt cgcctggtgg 900
ggcgccgagg aagccggcct ggtgggctcg acccactacg tgcagaacct cgccccggaa 960
gagaagaaga agatcaaggc ctacctgaac ttcgacatga tcggctcgcc gaacttcggc 1020
aacttcatct atgacggcga cggttccgac ttcggcctcc agggtccgcc cggctcggcc 1080
gccatcgagc gcctgttcga agcctacttc cgcctgcgcg gccagcaatc ggaaggcacc 1140
gagatcgact tccgctccga ctacgccgag ttcttcaaca gcggcatcgc cttcggcggc 1200
ctgttcaccg gcgccgaggg cctgaagacc gaagagcagg cgcagaagta cggcggcacc 1260
gccggcaagg cctacgacga gtgctaccac agcaagtgcg acggcatcgc caacatcaac 1320
caggacgccc tggagatcca cagcgacgcc atggccttcg tgaccagttg gctgtcgctg 1380
tcgaccaagg tggtcgacga cgagatcgcc gccgccggcc agaaagcaca atcgcggtcg 1440
ctgcagatgc agaagagcgc cagccagatc gaacgctggg gtcacgactt catcaagtaa 1500
Claims (13)
1. a kind of fermentation process of heat-resisting lysine aminopeptidase, it is characterised in that comprise the following steps：
(1) recombinant yeast pichia pastoris seed liquor is seeded to fermented and cultured in BSM culture mediums by 8-12% inoculum concentrations, the restructuring is finished
Red yeast is the Pichia yeast containing the lysine aminopeptidase gene from pseudomonas aeruginosa NJ-814, and the verdigris is false
Monad NJ-814 deposit number is CGMCC No.7638；
(2) after the initial glycerine in culture medium has consumed, glycerol adding supplemented medium is flowed, works as OD600Reach and stop after 350-450
Stream adds, and then stream plus methanol aqueous solution carry out Fiber differentiation, stop fermented and cultured when enzyme activity declines, obtain the heat-resisting bad ammonia
Sour aminopeptidase.
2. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (1),
Initial speed is 450-550rmin-1, throughput is fermented and cultured under conditions of 2vvm, 30 DEG C.
3. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), institute
The flow acceleration for stating glycerine supplemented medium is 17-19mLh-1·L-1。
4. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that in step (1), institute
The preparation method for stating recombinant yeast pichia pastoris seed liquor comprises the following steps：By the recombinant yeast pichia pastoris after activation in BMGY culture mediums
In, in 25-35 DEG C, 200-250rmin-1Rotating speed under cultivate.
5. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), stream
Plus also include the step of starvation is cultivated before methanol aqueous solution.
6. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), stream
Plus during methanol aqueous solution, in 1-2h, methanol aqueous solution flow acceleration is 1.0-1.5mLh-1·L-1, during 2h-3h, methanol-water
Solution flow acceleration is 1.5-2.5mLh-1·L-1, constant speed stream adds after 3h, and constant speed flow acceleration is 2.5-3.0mLh-1·
L-1。
7. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), institute
It is 45-55% glycerine and 1.0-1.5% PTM1 that glycerine supplemented medium, which is stated, including mass fraction.
8. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), first
Contain the PTM1 that mass fraction is 1.0-1.5% in alcohol solution.
9. the fermentation process of heat-resisting lysine aminopeptidase according to claim 1, it is characterised in that：In step (2), hair
Dissolved oxygen is 20-30% in ferment incubation.
10. the fermentation process of the heat-resisting lysine aminopeptidase according to any one of claim 1-9, it is characterised in that
After step (2), in addition to following purification step：
(S1) ammonium sulfate for being 40%-60% with mass fraction is saltoutd heat-resisting lysine aminopeptidase, is then dialysed；
(S2) anion-exchange chromatography is carried out to the product that dialysis is obtained；
(S3) the heat-resisting lysine aminopeptidase liquid that concentration, filtration chromatography are obtained, then through gel permeation chromatography.
11. the fermentation process of heat-resisting lysine aminopeptidase according to claim 10, it is characterised in that：In step (S2)
In, anion-exchange chromatography is carried out using Hitrap Q HP anion-exchange columns.
12. the fermentation process of heat-resisting lysine aminopeptidase according to claim 10, it is characterised in that：In step (S3)
In, the molecular cut off of the film used during filtering is 20-40kDa.
13. the fermentation process of heat-resisting lysine aminopeptidase according to claim 10, it is characterised in that：In step (S3)
In, gel permeation chromatography is carried out using the 10/300GL Filter columns of Superdex TM 75.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710237381.9A CN107129978A (en) | 2017-04-12 | 2017-04-12 | The fermentation process of heat-resisting lysine aminopeptidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710237381.9A CN107129978A (en) | 2017-04-12 | 2017-04-12 | The fermentation process of heat-resisting lysine aminopeptidase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107129978A true CN107129978A (en) | 2017-09-05 |
Family
ID=59716462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710237381.9A Pending CN107129978A (en) | 2017-04-12 | 2017-04-12 | The fermentation process of heat-resisting lysine aminopeptidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107129978A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107264A (en) * | 2021-12-07 | 2022-03-01 | 武汉新华扬生物股份有限公司 | Method for producing aminopeptidase by fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361287A (en) * | 2013-06-25 | 2013-10-23 | 江南大学 | Producing strain of heat-resisting aminopeptidase and purification method of heat-resisting aminopeptidase |
| CN104928315A (en) * | 2015-07-02 | 2015-09-23 | 江南大学 | Construction and expression method of recombinant pichia pastoris strain expressing lysine aminopeptidase |
| CN105002103A (en) * | 2015-06-06 | 2015-10-28 | 山东新时代药业有限公司 | Fermentation method for recombinant G-CSF(15-75) polypeptide pichia pastoris |
-
2017
- 2017-04-12 CN CN201710237381.9A patent/CN107129978A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361287A (en) * | 2013-06-25 | 2013-10-23 | 江南大学 | Producing strain of heat-resisting aminopeptidase and purification method of heat-resisting aminopeptidase |
| CN105002103A (en) * | 2015-06-06 | 2015-10-28 | 山东新时代药业有限公司 | Fermentation method for recombinant G-CSF(15-75) polypeptide pichia pastoris |
| CN104928315A (en) * | 2015-07-02 | 2015-09-23 | 江南大学 | Construction and expression method of recombinant pichia pastoris strain expressing lysine aminopeptidase |
Non-Patent Citations (2)
| Title |
|---|
| 刘强: "细菌耐热赖氨酸氨肽酶毕赤酵母表达及发酵优化", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 吴延涛等: "耐热赖氨酸氨肽酶菌株筛选、鉴定及基因克隆", 《食品与生物技术学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107264A (en) * | 2021-12-07 | 2022-03-01 | 武汉新华扬生物股份有限公司 | Method for producing aminopeptidase by fermentation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109370975B (en) | Method for improving yield of L-arginine synthesized by corynebacterium crenatum | |
| CN110777136B (en) | Alkaline protease mutant for washing and application thereof in liquid detergent | |
| CN103074315B (en) | Lipase LIP, gene and application thereof | |
| CN107325977B (en) | Method for producing protein glutaminase, purification and application | |
| CN105695340B (en) | Aspergillus oryzae and application thereof | |
| CN104726478A (en) | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli | |
| CN111218437A (en) | High-yield alkaline lipase, gene, strain and application | |
| CN105002103B (en) | A kind of recombination G-CSF（15-75）The fermentation process of polypeptide Pichia yeast | |
| CN110343691A (en) | Saltant type heparinase I and its coding nucleotide sequence, the recombinant vector including the nucleotide sequence and host cell and application | |
| CN111777680B (en) | Separation and purification process for improving stability of recombinant collagen solution | |
| CN107129978A (en) | The fermentation process of heat-resisting lysine aminopeptidase | |
| CN104278017A (en) | Recombinant expression method of human lysozyme | |
| CN112877227B (en) | Pichia pastoris strain for high yield of rhamnosidase | |
| CN114703165B (en) | Beta-glucosidase mutant and application thereof | |
| CN107778365A (en) | The recombinant-protein expression that a kind of multiple spot is integrated | |
| CN109957538A (en) | A kind of genetic engineering bacterium and its preparation method and application preparing sarcosine oxidase | |
| CN105296448A (en) | Agarase and preparation method thereof | |
| CN113564151B (en) | Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof | |
| CN107325978B (en) | Protein glutaminase producing strain, preparation and application of fermentation product | |
| CN109161556A (en) | M1PDH gene, its protein and purposes in one main laminaria | |
| CN105420220B (en) | A kind of aspartic acid albuminoid enzyme and its encoding gene and application | |
| CN116179582A (en) | Preparation method and application of universal gene recombinant protein A affinity purification filler | |
| CN114908074A (en) | Aqueous solution stable alkaline protease 2709 mutant and preparation method and application thereof | |
| CN103773793B (en) | A kind of method of high efficient expression human serum albumins | |
| CN101121747A (en) | Method for separating and purifying glutamine transaminase activation proteinase inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170905 |
|
| RJ01 | Rejection of invention patent application after publication |