CN103333879A - Method for immobilizing marine lipase Bohai Sea-9145 with magnetic Fe3O4@SiO2-NH2 nanoparticles - Google Patents

Method for immobilizing marine lipase Bohai Sea-9145 with magnetic Fe3O4@SiO2-NH2 nanoparticles Download PDF

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CN103333879A
CN103333879A CN2013102540781A CN201310254078A CN103333879A CN 103333879 A CN103333879 A CN 103333879A CN 2013102540781 A CN2013102540781 A CN 2013102540781A CN 201310254078 A CN201310254078 A CN 201310254078A CN 103333879 A CN103333879 A CN 103333879A
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lipase
immobilized
enzyme
marine
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孙谧
徐甲坤
盛军
郑鸿飞
刘均忠
琚彩霞
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

The invention relates to a method for immobilizing marine lipase Bohai Sea-9145 with magnetic Fe3O4@SiO2-NH2 nanoparticles. The method is characterized by comprising the following steps of: putting the magnetic Fe3O4@SiO2-NH2 nanoparticles in a reactor, then adding a marine lipase Bohai Sea-9145 solution with enzyme protein concentration of 0.0375-0.225mg/ml and pH value of 4.0-10.0 to the reactor to implement an immobilization reaction for 4-20 hours at 20-50 DEG C, then carrying out magnetic separation, collecting the supernatant for determining the content of the protein, and washing immobilized marine lipase Bohai Sea-9145 until no protein content in the washing solution is absorbed, thereby obtaining immobilized marine lipase Bohai Sea-9145. The method has the characteristics that the enzyme activity stability promotion effect of the immobilized marine lipase Bohai Sea-9145 is obvious, the enzyme activity recovery rate is as high as 87.7%, the obtained immobilized enzyme has good reutilization property and high mechanical strength and can still keep higher lipase activity after being stored for a long time, and the method is an ideal enzyme immobilization method; and therefore the method is more widely applied to industries, medicines, biochemical analysis and the like.

Description

Magnetic Fe 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145
Technical field
The present invention relates to the marine microorganism field, particularly relate to magnetic Fe 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145.
Background technology
Lipase (EC3.1.1.3) is the carboxyl lytic enzyme of the inside and outside glycerine of the main catalysis cell of a class and long-chain fat acid hydrolysis, ester synthesis reaction, is a kind of interfacial enzymes that degradation of substrates is formed free fatty acids and glycerine by reaction.It belongs to a class lytic enzyme (E.C.3), acts on the ester bond (E.C.3.1.1) of carboxylicesters (E.C.3.1), can hydrolyzing triglyceride, and triglyceride and monoglyceride, and be called triacylglycerol Acyl-hydrolase (E.C.3.1.1.3).The catalyzed reaction of lipase is in extensive range, can catalysis comprise hydrolysis, transesterify, and alcoholysis, acidolysis, esterification and ammonia solution are at interior multiple biochemical reaction.Lipase can both play to multiple substrate that regioselectivity is synthetic, the effect of enantiomorph catalysis.
The lipase that cryophile produces in the ocean environment can carry out catalytic hydrolysis reaction at low temperatures efficiently, and common have a liking for warm microorganism only can show seldom activity under cold condition.These lipase have given low-temperature lipase low enthalpy of activation in the singularity that structure function possesses higher handiness, particularly its avtive spot, the high specific acitivity under low substrate avidity and the low temperature.
The resolvase catalyzed reaction is carried out in solution, poor stability in general, easily inactivation, be insoluble to organic solvent, be subject to the influence of factors such as environment.Immobilized enzyme is by appropriate means enzyme to be fixed in certain space, can realize reclaiming and recycling when keeping himself characteristic.The appearance of immobilized enzyme has solved the many limitations in the resolvase practical application, the resolvase less stable, and problems such as lipase separation and purification difficulty have limited the utilization of ocean lipase.
Summary of the invention
The objective of the invention is to overcome above-mentioned prior art and have many deficiencies, through the long-term development research marine microorganism of contriver and enzyme product and production practice, exploitation provides a kind of immobilization system stable, and the enzyme motility rate keeps high magnetic Fe 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145 comprises the following steps:
With magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere places container (as cillin bottle), adding zymoprotein concentration in the container again is the ocean lipase B ohai Sea-9145 solution of 0.0375 1 0.225mg/ml, pH4.0-10.0, carry out immobilized reactant at shaking table, the immobilized reactant temperature is 20-50 ℃, the immobilized reactant time is 4-20 hour, carry out magnetic resolution then, collect supernatant liquor, be used for measuring protein content, clean immobilized marine lipase B ohai Sea-9145, no protein content absorbs in scavenging solution, gets immobilized marine lipase B ohai Sea-9145.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohai Sea-9145, the numbering of described ocean lipase B ohai Sea-9145(ocean lipase B ohai Sea-9145 is to distinguish other low-temperature alkali lipase, hereinafter referred lipase B ohai Sea-9145 or lipase) be the lipase that is produced by suitable to cold marine yeast bacterial strain Bohai Sea-9145 fermentation culture, be a kind of marine low temperature lipase.This ocean lipase B ohai Sea-9145 has stronger hydrolytic action for the ester class substrate of middle long-chain, can keep lipase activity preferably in low temperature environment, and action pH and temperature range are wider simultaneously, demonstrate good enzymatic property.
Described suitable to cold marine yeast bacterial strain Bohai Sea-9145 is the high-yield of low-temperature alkaline lipase Bohai Sea-9145(ocean lipase B ohai Sea-9145 that separate to obtain from the ooze of marine site, the Bohai Sea) suitable to cold marine yeast bacterial strain Bohai Sea-9145, according to the 4th edition " The Yeasts, A Taxonmic study ", (Kurtzman﹠amp; Fell, 1998) be defined as the suitable to cold ocean and separate new subspecies of fat Luo Shi yeast strain (Yarrowia lipolytica strain), this suitable to cold marine yeast bacterial strain Bohai Sea-9145 is preserved in Chinese China typical culture collection center, Wuhan (being called for short CCTCC) on November 26th, 2003, and preserving number is CCTCCNO:M203095.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohai Sea-9145, ocean lipase B ohai Sea-9145 by Huanghai Sea aquatic products institute enzyme engineering chamber by following fermentation culture gained.
Produce the fermentation culture of enzyme suitable to cold marine yeast bacterial strain BohaiSea-9145
Suitable to cold marine yeast bacterial strain BohaiSea-9145 was cultivated 24 hours in 25 ℃ on biochemical incubator middle plateform, and substratum (wt%) is peptone 3%, beef extract 2%, KH 2PO 40.2%, MgSO 40.05%, sweet oil 0.5% is with Na 2HPO 4Adjusting pH is 6.5-7.0, obtains bacterium colony cheese shape, creamy white, and not reflective, the edge is wavy, and tree root shape projection is arranged.And on industrial shaking table in pH5.0, inoculum size 6% is planted 21h in age, the 250ml triangle shakes bottled amount 50ml, it is 4% soybean cake powder that the carbon nitrogen of substratum is formed (wt%), 4% peanut meal, 4% rice bran and 0.5 rough peanut oil, MgSO 40.05%, KH 2PO 40.2%, pH5.0 in 26 ± 1 ℃ of inoculation culture 23 hours, gets fermented liquid, with fermented supernatant fluid, adds trichloromethane (VCH 3CL:V supernatant liquor=5:100) fully stir left standstill 30 fens in 4 ℃, and with 4000rpm centrifugal 40 minutes, to remove foreign protein and also collect supernatant liquor, this lipase is up to 15828u/g than living, and purity improves 1.2.Get the extraction supernatant liquor and carry out ultrafiltration through the 100KDa hollow fiber column, concentrate with the 10KDa hollow fiber column, lipase is 41288 than work, improves 3.3 times.And carry out chromatography with CM Sepharose FF cation-exchange chromatography post, substep is collected elutriant, measure its low-temperature alkali lipase and be that than living 206224u/g, purity improve 5.0 times of (mensuration of lipase activity: adopt People's Republic of China's industry standard (Ministry of Light Industry of the People's Republic of China (PRC) 1993).The hydrolysis unit of activity of lipase is defined as: with lipase hydrolyzation of oil and fat, per minute produces the enzyme amount of 1 μ mol lipid acid, is defined as a lipase activity unit of force).
Fermentation culture by above-mentioned bacterium producing multi enzyme preparation obtains ocean lipase B ohai Sea-9145, its molecular weight obtains electrophoretically pure low-temperature alkali lipase through CM Sepharose purifying, analyze by the Laemmli method with SDS-PAGE and to be single protein band, molecular weight is 38Kda; 35 ℃ of optimal reactive temperatures, optimal pH are 8.5; Equilibrium temperature is lower than 35 ℃, and stablizing the PH scope is 4.0-9.0; With common metal ion compatibleness:
Figure 2013102540781100002DEST_PATH_IMAGE001
Zn 2+The ion pair low-temperature alkali lipase has reversible rejection characteristic (more than see Chinese patent ZL200410096668.7 for details).
Suitable to cold marine yeast bacterial strain provided by the invention (Yarrowia lipolytica) BohaiSea-9145 is the superior strain with low-temperature alkali lipase of good commercial application prospect, fermentation period is short, produce the enzyme temperature-stable, raw material sources are extensive, low production cost is conducive to further open up the Application Areas of marine microorganism resource.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohaiSea-9145, described magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere is synthetic by following method:
In a container (as round-bottomed flask), add certain water gaging (like the container size), the reaction system argon shield, by means of the liquid nitrogen multigelation, vacuum degassing process will be by the FeCl of stoichiometric quantity except the oxygen in anhydrating 24H 2O and FeCl 36H 2O is in container, and argon shield is heated to 80 ℃, behind the question response system homogeneous; dropwise add the 29wt%50ml ammonia soln and become black, continue heating 20min, magnetic absorption; be neutral to washings for several times with distilled water and dehydrated alcohol repetitive scrubbing respectively afterwards, evaporate to dryness gets black magnetic Fe 3O 4Nano microsphere (MNP).
Magnetic Fe 3O 4@SiO 2Synthesizing of Nano microsphere:
With magnetic Fe 3O 4Nano microsphere 1.0g is in two mouthfuls of flasks of 250ml, add the 80ml dehydrated alcohol, and 20ml water, it is even to system to stir 10min under the ar gas environment, ultrasonic 10min, dropwise add ammoniacal liquor 1.6ml to pH9.0 under the magnetic agitation, ultrasonic tetraethoxy (TEOS) 2.0ml that adds down, 40 ℃ of reaction 4h, magnetic absorption, difference water and absolute ethanol washing product several, evaporate to dryness gets magnetic Fe 3O 4@SiO 2Nano microsphere (SMNP).
Magnetic Fe 3O 4@SiO 2-NH 2Synthesizing of Nano microsphere (AMNP):
Again with magnetic Fe 3O 4@SiO 2Nano microsphere 10mg is in two mouthfuls of flasks of 100ml; pipette 20ml toluene in two mouthfuls of flasks; argon shield; magnetic agitation is disperseed magnetic nano-particle in toluene solution, dropwise add 1.0ml3-aminopropyl triethoxysilane (APTES), magnetic agitation reaction 24h; magnetic absorption; with absolute ethanol washing for several times, evaporate to dryness gets magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere (AMNP).
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohaiSea-9145, the immobilized marine lipase B ohaiSea-9145 reuse that obtains is good, the physical strength height still can keep higher lipase activity through long-time storage, is a kind of desirable immobilized enzyme method.Therefore, have application space widely at aspects such as industry, medicine, biochemical analysises.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohaiSea-9145:
(1) zymoprotein concentration is to the influence of immobilization effect
In immobilized marine lipase B ohaiSea-9145, work as magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere (carrier) content just can reach optimal effect when consistent with protein content in the immobilized enzyme liquid.Shown in Figure 1, increase along with zymoprotein concentration, protein adsorption quantity also increases, zymoprotein concentration from 0.0375mg/ml to 0.125mg/ml between, it is obvious that the protein adsorption rate increases trend comparison, explanation, when zymoprotein concentration hour, the immobilization of unit carrier does not occur saturated, and therefore along with zymoprotein concentration increases, adsorption rate increases and increases; And zymoprotein concentration is during from 0.125mg/ml to 0.225mg/ml, and along with zymoprotein concentration increases, adsorption rate still has the trend of increase, but curve becomes gently, illustrates, when zymoprotein concentration during at 0.125mg/ml, the protein adsorption rate is near saturated.This moment, the immobilized lipase enzymatic activity recovery reached maximum value, though zymoprotein concentration is during from 0.125mg/ml to 0.225mg/ml, along with zymoprotein concentration increases, unit carrier zymoprotein adsorptive capacity has the trend of increase, the vigor of immobilized enzyme but presents the trend of reduction, but the unit's of explanation carrier enzyme immobilization amount is limited, when zymoprotein concentration is low, along with the increase of zymoprotein concentration, active corresponding raising, but when enzyme liquid concentration reached certain value, the enzyme molecule was assembled mutually and is caused enzyme molecular activity center to hide mutually, influences the enzyme-to-substrate combination, thereby make active the reduction, shown in Figure 2, so zymoprotein concentration is preferably 0.125mg/ml.
(2) immobilization temperature is to the influence of immobilization effect
Be under 20 ℃ of-50 ℃ of conditions, to the influence of protein adsorption rate, as can be seen from Figure 3 in immobilization (reaction) temperature, rising along with temperature, the protein adsorption rate slowly increases, and changes not quite, illustrates that the immobilization temperature is little to the influence of unit carrier proteins adsorptive capacity.As shown in Figure 4, along with the continuous rising of temperature of reaction, the immobilized enzyme vigor presents the trend of slow increase, temperature of reaction reaches maximum value when being 30 ℃, along with temperature raises, the protein adsorption rate slowly increases, but the immobilized enzyme enzyme is lived and is reduced gradually, illustrates, easily makes the zymoprotein inactivation under the high temperature.So the immobilization temperature is preferably 30 ℃.
(3) pH value of reaction system is to the influence of immobilization effect
Enzyme and carrier are at different pH(pH4.0-12.0) different dissociated states is arranged under the condition, thereby immobilization effect (protein adsorption rate and immobilized enzyme vigor thereof) is also had influence in various degree.As can be seen from Figure 5, along with the increase of system pH, the protein adsorption rate presents elder generation and increases the trend that afterwards reduces, and when the pH value was 8.0, adsorption rate reached maximum value, was the 22.65mg/g carrier.As can be seen from Figure 6, be 8.0 o'clock in the pH value, the vigor of immobilized enzyme is the highest, and along with the enhancing of acid/alkalescence, vigor all weakens gradually, so pH value of reaction system is 8.0.
Above-mentioned different acidity damping fluid compound method all is well known in the art: at this, pH4.0-12.0 of the present invention is by following preparation (being 50mM):
PH4.0: take by weighing 0.612g NaAc3H 2O, the HAc of absorption 1.025ml, adding distil water dissolving and constant volume are to 500ml;
PH5.0: take by weighing 2.381g NaAc3H 2O, the HAc of absorption 0.375ml, adding distil water dissolving and constant volume are to 500ml;
PH6.0: take by weighing the 0.436g anhydrous Na 2HPO 4With 3.421g NaH 2PO 42H 2O, adding distil water dissolving and constant volume are to 500ml;
PH7.0: take by weighing the 2.166g anhydrous Na 2HPO 4With 1.521g NaH 2PO 42H 2O, adding distil water dissolving and constant volume are to 500ml;
PH8.0: take by weighing the 3.362g anhydrous Na 2HPO 4With 0.206g NaH 2PO 42H 2O, adding distil water dissolving and constant volume are to 500ml;
PH9.0: take by weighing 1.876g glycine and 0.176g NaOH, adding distil water dissolving and constant volume are to 500ml;
PH10.0: take by weighing 1.876g glycine and 0.652g NaOH, adding distil water dissolving and constant volume are to 500ml;
PH11.0: take by weighing 4.475g Na 2HPO 412H 2O and 0.08g NaOH, adding distil water dissolving and constant volume are to 500ml;
PH12.0: take by weighing 0.466gKCl and 0.06g NaOH, adding distil water dissolving and constant volume are to 500ml.
(4) the immobilization time is to the influence of immobilization effect
The immobilization time to the influence of zymoprotein adsorption rate and immobilized lipase enzyme activity as shown in Figure 7, when the immobilization time is 4-10h, unit carrier proteins adsorptive capacity rises very fast, this is because there is a large amount of relatively resolvase molecules in immobilized reactant at the beginning in the reaction system, immobilized reactant speed is very fast, after the immobilized reactant time reached 10h, protein adsorption was little big, but changes not obvious.The expression immobilization time is to the influence of immobilized enzyme vigor among Fig. 8, the immobilized reactant time is in the 2-5h scope, the immobilized enzyme vigor improves constantly, this be because, along with continuing of reaction times, the protein adsorption rate of carrier constantly increases, so immobilized enzyme work presents continuous increase tendency, but be in the 10-20h scope time when the immobilization time, and the immobilized enzyme vigor descend to some extent, this shows that the best immobilization time is 10h.So the immobilization time is preferably 10h.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohaiSea-9145, preferred fixing condition is: zymoprotein concentration is 0.125mg/ml, the immobilization temperature is 30 ℃, the pH value of immobilization system is 8.0, the immobilization time is 10h, enzymatic activity recovery can reach 87.7%, and the protein adsorption rate of unit surface carrier can reach 23.7mg/g.
Magnetic Fe provided by the invention 3O 4@SiO 2-NH 2In the method for Nano microsphere immobilized marine lipase B ohaiSea-9145, immobilized marine lipase B ohaiSea-9145(is called for short immobilized enzyme) zymologic property along with the immobilization of enzyme molecule after, the variation of its state, corresponding variation also can take place simultaneously in zymologic property, the zymologic property of immobilized marine lipase B ohaiSea-9145 mainly shows the vigor of immobilized enzyme, the stability of immobilized enzyme and the response characteristic of immobilized enzyme, on the basis to the research of ocean lipase B ohaiSea-9145 fixing condition, zymologic property to immobilized marine lipase B ohaiSea-9145 is studied, and compare with resolvase (not immobilized enzyme) zymologic property, provide reliable theoretical foundation in the hope of using for the production of immobilized lipase.
The immobilized enzyme optimal reactive temperature:
With the immobilized ocean of preferred fixing condition lipase B ohaiSea-9145, the vigor of immobilized lipase and free-fat enzyme changes under the research differing temps.Resolvase and the vigor of immobilized enzyme under differing temps in 20 ℃～90 ℃ scopes, have been measured respectively, experimental result shows as shown in Figure 9, the optimal reactive temperature of immobilized enzyme and resolvase is respectively 40 ℃ and 35 ℃, wherein, from 20 ℃ to 38 ℃, resolvase more can have been given play to bigger vigor than immobilized lipase, resolvase is described, and its catalytic capability temperature influence is less at a lower temperature, and the vigor that immobilized enzyme shows in lower temperature range is lower, show that immobilized enzyme vigor temperature influence at a lower temperature is then bigger.
Along with the rising of temperature of reaction system, can activating enzyme and the speed of substrate reactions.For resolvase, in the time of 35 ℃, its vigor reaches maximum value, and relative vigor reduces to 64% in the time of 50 ℃, and then along with temperature raises, the resolvase vigor reduces rapidly, and activity completely loses in the time of 70 ℃.Immobilized enzyme vigor in the time of 40 ℃ reaches maximum value, illustrate that 40 ℃ are its optimal reactive temperature, when temperature is in 40 ℃ of-50 ℃ of scopes, the immobilized enzyme vigor slowly reduces, in the time of 70 ℃, the relative enzyme activity of immobilized enzyme vigor is 20 ℃, 80 ℃ of loss of activity, illustrates that resolvase immobilization rear stability has obtained certain raising.
Immobilized enzyme optimal reaction pH value:
Obtain lipase B ohaiSea-9145 with preferred fixing condition immobilization, in being the substrate solution of 4.0-10.0, pH carries out catalyzed reaction respectively, the situation that immobilized lipase and free-fat enzyme activity change under the exploration condition of different pH, experimental result shows as shown in figure 10, the optimal reaction pH value of immobilized enzyme and resolvase is 8.0, shows that immobilization process is little to the optimal reaction pH value influence of resolvase.System pH is 4.0 o'clock, the resolvase complete deactivation, rising along with system pH, the resolvase vigor increases gradually, system pH is that 8.0 o'clock vigor reach maximum value, and the back is along with the enhancing of reaction system alkalescence, and the resolvase vigor is reduction trend, when system pH was 10.0, enzyme activity was 65% relatively; For resolvase, the immobilized enzyme acid-fast alkali-proof has obtained certain raising, and when the pH value was 4.0, its relative enzyme activity was 26.31%, along with pH in the system increases, the immobilized enzyme vigor increases gradually, is 8.0 o'clock in the pH value, and the immobilized enzyme vigor reaches maximum value, then along with the increase of system pH, the immobilized enzyme vigor reduces gradually, reaches at 10.0 o'clock in the pH value, and the relative enzyme activity of immobilized enzyme reduces to 80%.
The thermostability of immobilized enzyme and resolvase:
The PBS(pH acidity damping fluid of immobilized lipase and free-fat enzyme) (pH8.0's solution 50mM) lowers the temperature in ice bath behind the child care 60min in 20～60 ℃ of water-baths respectively, investigates its vigor and changes experimental result such as Figure 11.Immobilized enzyme and resolvase are that 20～40 ℃ of inner enzyme vigors are comparatively stable in reaction system, and 50 ℃ of enzyme activities begin decline in various degree.Resolvase behind 40 ℃ of reaction 60min, relatively enzyme activity be behind 72%, 50 ℃ of reaction 60min relatively enzyme activity be behind 31%, 60 ℃ of reaction 60min relatively enzyme activity only have 10%; Behind 40 ℃ in the immobilized enzyme reaction 60min relatively enzyme activity still have 91%, 50 ℃ down behind the reaction 60min, enzyme activity is 72%, 60 ℃ down behind the reaction 60min relatively, enzyme activity still has 42% relatively.Show thus, immobilization process, improved the stability of enzyme significantly, this is because the enzyme immobilization process relates between the zymoprotein molecule and the interaction between enzyme and the carrier, this makes the structure rigidity of enzyme molecule strengthen, thereby its thermostability is greatly improved, and resolvase is easier to the sex change inactivation owing to lacking these reactive forces.
The pH stability of immobilized enzyme and resolvase:
With immobilized lipase and free-fat enzyme respectively in the corresponding buffer solution system of pH4.0-12.0 in 30 ℃ of insulation 24h, measure its corresponding enzyme activity respectively, investigate the pH stability of enzyme.With the full-page proof of enzyme activity sample in contrast, hence one can see that as shown in figure 12 for the result, immobilized enzyme vigor stable range is pH6.0-10.0, pH is that 9.0 o'clock stability is best, the residual enzyme vigor still keeps 94%, resolvase stability when pH9.0 is best, and the residual enzyme vigor is 56%, illustrates that the resistance to acids and bases of immobilized enzyme is apparently higher than resolvase.
The reusability of immobilized enzyme:
With immobilized lipase and substrate p-nitrophenyl laurate in 40 ℃ of catalyzed reaction 8min, after reaction finishes, magnetic resolution, collect supernatant liquor, be used for measuring the vigor of its immobilized enzyme, the immobilized enzyme after magnetic separates is used further to the catalyzed reaction under repetition the same terms, and the like, catalysis is 8 batches altogether, measures each vigor that finishes the back enzyme respectively.Be 100% with first immobilized enzyme vigor, calculate the relative percentage that each reaction finishes back immobilized enzyme vigor, to analyze the reusability of immobilized lipase.
Experimental result as shown in figure 13.From figure as can be seen, when immobilized enzyme carries out catalyzed reaction for the second time, its vigor obviously descends, approximately reduce by 24%, this may be because after catalyzed reaction finishes for the first time, magnetic separates the loss that not quite causes part magnetic immobilized enzyme fully in the washing process, and when repeating catalyzed reaction eight times, the immobilized enzyme vigor still remains 44%.
According to transformation period calculation formula: t 1/2=0.693t/2.303lg (E 0/ E) [94](wherein, t 1/2Be the immobilized enzyme operation transformation period, t is for repeating access times, E 0Be initial enzyme activity, E is for reusing the back enzyme activity t time), calculating the immobilized enzyme operation transformation period is seven times, also illustrate, in the immobilization process, enzyme and carrier fine in conjunction with effect.Experimental result shows that the reusability of immobilized enzyme is better, simultaneously, also explanation, in the immobilization process, enzyme and carrier fine in conjunction with effect.
The stability in storage of immobilized enzyme:
(pH9.0 50mM) placed respectively 5-30 days under 25 ℃ of conditions, measured enzyme respectively and lived and calculate the enzyme relative percentage of living, to analyze its stability in storage with the PBS solution of immobilized lipase and free-fat enzyme.
The result as shown in figure 14, its stability in storage is significantly increased behind the enzyme immobilization, stores 30 days, resolvase keeps 59% vigor, and the relative reactivity of immobilized enzyme still can keep 81%.Utilize the storage transformation period under 25 ℃ of transformation period being fixed of calculation formula enzyme and the resolvases to be respectively 99 days and 39 days.
The apparent Michaelis-Menton constant K of immobilized enzyme and resolvase mPH-value determination pH:
This experiment is selected at 40 ℃, pH8.0, in the middle of the PBS damping fluid of 50mM, select different concns p-nitrophenyl laurate as substrate, measure free-fat enzyme and immobilized lipase its speed of response under different substrate-functions, then with the 1/[S of 1/V] make the Lineweaver-Burk curve, pass through formula:
1/V=(K m/ V Max) (1/[S])+1/V Max, try to achieve Km, Vmax.By formula (1/V=(K m/ V Max) (1/[S])+1/V Max) as can be known, slope is Km/Vmax in the resulting curve, intercept is 1/Vmax, so Km equals slope/intercept, Vmax is 1/ intercept, calculates gained free-fat enzyme and immobilized lipase
The kinetic parameter result is as follows:
Figure BDA00003395849900091
Data presentation, the Michaelis-Menton constant Km of immobilized lipase is elevated to 0.79mg/ml by 0.23mg/ml, and corresponding maximum initial velocity of reaction Vm then is reduced to 40.98mg/minml by the 75.53mg/minml of resolvase.Lipase is through after the immobilization, and its substrate avidity obviously descends.
This shows magnetic Fe provided by the invention 3O 4@SiO 2-NH 2The immobilized marine lipase B ohai Sea-91451 reuse that the method for Nano microsphere immobilized marine lipase B ohai Sea-9145 obtains is good, the physical strength height, still can keep higher lipase activity through long-time storage, be a kind of desirable immobilized enzyme method.Therefore have application space widely at aspects such as industry, medicine, biochemical analysises.
Preparation method's characteristics of the suitable cold yeast Bohai Sea-9145 lipase B ohai Sea-9145 of magnetic Nano material immobilized marine provided by the invention are:
1. suitable to cold marine yeast bacterial strain provided by the invention (Yarrowia lipolytica) BohaiSea-9145 is the low-temperature alkali lipase superior strain with good commercial application prospect, fermentation period is short, produce the enzyme temperature-stable, raw material sources are extensive, low production cost is conducive to further open up the Application Areas of marine microorganism resource.
2. this ocean lipase B ohai Sea-9145 has stronger hydrolytic action for the ester class substrate of middle long-chain, can keep lipase activity preferably in low temperature environment, and action pH and temperature range are wider simultaneously, demonstrate good enzymatic property.The immobilized enzyme reuse is good, and the physical strength height still can keep higher lipase activity through long-time storage, is a kind of desirable immobilized enzyme method.Therefore have application space widely at aspects such as industry, medicine, biochemical analysises.
3. obtain the maximum flexibility enzymatic activity recovery after the immobilized marine lipase B ohai Sea-9145 immobilization and can reach 87.7%, the protein adsorption rate of unit surface carrier can reach 23.7mg/g.
Immobilized marine lipase B ohai Sea-9145 provided by the invention has application space widely at aspects such as industry, medicine, biochemical analysises.
Description of drawings
The different zymoprotein concentration of Fig. 1 are to the influence of adsorptive capacity
The different zymoprotein concentration of Fig. 2 are to the influence of immobilized lipase enzyme activity
Fig. 3 immobilization temperature is to the influence of adsorption rate
Fig. 4 immobilization temperature is to the influence of immobilized lipase enzyme activity
Fig. 5 system pH is to the influence of protein adsorption rate
Fig. 6 system pH is to the influence of immobilized enzyme vigor
Fig. 7 immobilization time is to the influence of protein adsorption rate
Fig. 8 immobilization time is to the influence of immobilized enzyme vigor
Fig. 9 temperature is to the influence of resolvase and immobilized enzyme vigor
The influence of the resolvase of Figure 10 pH and immobilized enzyme vigor
The thermostability of Figure 11 resolvase and immobilized enzyme
The pH stability of Figure 12 resolvase and immobilized enzyme
The reusability of Figure 13 immobilized lipase
The storage stability of Figure 14 resolvase and immobilized lipase
The kinetic curve of Figure 15 free-fat enzyme and immobilized lipase
Embodiment
The present invention further specifies the present invention with the following example, but protection scope of the present invention is not limited to the following example.
Embodiment 1
With the 10mg magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere places the 10ml cillin bottle, adding zymoprotein concentration in the cillin bottle again is 0.125, mg/ml, the pH of immobilization system is 8.0 lipase solution 2ml, the immobilized reactant temperature is 30 ℃, between shaking table carries out immobilized reactant 10 hours, carry out magnetic resolution then, collect supernatant, be used for measuring protein content, after immobilized enzyme cleans for several times, no protein content absorbs in supernatant liquor, and enzyme is used for measuring immobilized enzyme lives, and gets immobilized marine lipase B ohai Sea-9145, enzymatic activity recovery is 87.7%, and the protein adsorption rate of unit surface carrier is 23.7mg/g.
Embodiment 2
With the 10mg magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere places the 10ml cillin bottle, adding zymoprotein concentration in the cillin bottle again is 0.150, mg/ml, the pH of immobilization system is 7.0 lipase solution 2ml, the immobilized reactant temperature is 25 ℃, between shaking table carries out immobilized reactant 12 hours, carry out magnetic resolution then, collect supernatant, be used for measuring protein content, after immobilized enzyme cleans for several times, no protein content absorbs in supernatant liquor, and enzyme is used for measuring immobilized enzyme lives, and gets immobilized marine lipase B ohai Sea-9145, enzymatic activity recovery is 86.5%, and the protein adsorption rate of unit surface carrier is 24.0mg/g.
Embodiment 3
With the 10mg magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere is in the 10ml cillin bottle, in cillin bottle, add zymoprotein concentration 0.1mg/ml, the pH of immobilization system is 7.0 lipase solution 2ml, the immobilized reactant temperature is 20 ℃, between shaking table carries out immobilized reactant 14 hours, carry out magnetic resolution then, collect supernatant, be used for measuring protein content, after immobilized enzyme cleans for several times, no protein content absorbs in supernatant liquor, and enzyme is used for measuring immobilized enzyme lives, and gets immobilized marine lipase B ohai Sea-9145, enzymatic activity recovery is 81.0%, and the protein adsorption rate of unit surface carrier is 22.0mg/g.

Claims (2)

1. magnetic Fe 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145 is characterized in that:
With magnetic Fe 3O 4@SiO 2-NH 2Nano microsphere places reactor, adding zymoprotein concentration in the container again is the ocean lipase B ohai Sea-9145 solution of 0.0375 1 0.225mg/ml, pH4.0-10.0, carry out immobilized reactant, the immobilized reactant temperature is 20-50 ℃, the immobilized reactant time is 4-20 hour, carry out magnetic resolution then, collect supernatant liquor and be used for measuring protein content, clean immobilized marine lipase B ohai Sea-9145, after no protein content absorbs in scavenging solution, get immobilized marine lipase B ohai Sea-9145.
2. according to the magnetic Fe of claim 1 3O 4@SiO 2-NH 2The method of Nano microsphere immobilized marine lipase B ohai Sea-9145 is characterized in that described protease concentration is 0.125mg/ml, and described immobilized reactant temperature is 30 ℃, and described pH value is 8.0, and the described immobilization time is 10h.
CN2013102540781A 2013-06-24 2013-06-24 Method for immobilizing marine lipase Bohai Sea-9145 with magnetic Fe3O4@SiO2-NH2 nanoparticles Pending CN103333879A (en)

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