CN104140961A - Immobilized lipase having Sn-1,3 specificity as well as preparation method and application of immobilized lipase - Google Patents
Immobilized lipase having Sn-1,3 specificity as well as preparation method and application of immobilized lipase Download PDFInfo
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Abstract
The invention discloses an immobilized lipase having a Sn-1,3 specificity as well as a preparation method and an application of the immobilized lipase. The preparation method comprises the following steps of dissolving the immobilized lipase having the Sn-1,3 specificity in a buffer solution of which the pH value is 9.0-10.0 to obtain an enzyme liquid; pre-treating resin, placing in the enzyme liquid for adsorbing, after the adsorption is completed, washing and drying to obtain the immobilized lipase having the Sn-1,3 specificity; the resin is styrene macroporous adsorbent resin of which the specific surface area is 200-600m<2>/g and the average pore diameter is shown in the specification. The invention also discloses the immobilized lipase having the Sn-1,3 specificity prepared by the preparation method and the application thereof. The activity of the immobilized lipase prepared by the prepared method disclosed by the invention, compared with that of a free lipase, is significantly improved, the Sn-1,3 specificity is maintained to be stable and the reaction time is shortened when the immobilized lipase is applied in synthetic reaction of OPO.
Description
Technical field
The present invention relates to human milk fat substituted product field, relate in particular to a kind of immobilization Sn-1,3 specific lipases and its preparation method and application.
Background technology
OPO (1,3-Dioleoyl-2-palmitoyl triglyceride, OPO) is a kind of nutrition-fortifying agent that China's new approval in 2008 is used, and is mainly used in dispensed food for baby.GB14880-2012 < < food safety national standard food enrichment is used in standard > >, also lists OPO in food enrichment.The preparation of OPO mainly synthesizes master with lipase-catalyzed transesterify at present.
Publication number is that the Chinese patent literature of CN101273118A discloses the method that employing oleic acid or its ester and triglyceride level catalyze and synthesize OPO.But its catalyzer adopting is generally commercially available immobilized lipase, because its price is compared with the high large-scale application of this type of immobilized lipase in oil prodution industry that limited.Therefore, preparation is suitable for requirement and immobilized lipase with low cost synthesizes the research emphasis that OPO becomes association area.
The immobilization of lipase, is with solid material, lipase is fettered or is limited in certain area, but still can guarantee the method for its distinctive catalytic activity.Immobilized lipase has the superiority that resolvase can not be compared,, enzyme high such as catalytic efficiency can reuse and then reduce costs, easily and product separation, improved enzyme stability, extended enzyme work-ing life, can realize continuous operation etc.
The ultimate principle of macroporous adsorbent resin immobilized lipase is absorption, and this method reaction conditions is gentle, less to enzyme molecular structure destructiveness, and enzyme is lived and is difficult for loss, and the specificity of enzyme is difficult for influenced.And macroporous adsorbent resin self structure is simple, material safety, pre-treatment is easy, is suitable for the immobilization of lipase in oil prodution industry.
The Sn-1 in aspergillus oryzae source, 3 specific lipase securities are good, in the acidolysis reaction of catalyzing glycerol three esters and lipid acid, have higher regiospecificity, can be specifically for triglyceride level Sn-1, acidolysis or transesterify are carried out in 3 positions, directed synthetic desired substance (as OPO).
Publication number is the preparation method that the Chinese patent literature of CN101280297A discloses a kind of immobilized lipase, concrete steps are: macroporous resin after pretreatment, add lipase liquid and microsphere, vibration absorption, after lyophilize, carry out interface activation technique processing, filtering and removing activator (heptane), lyophilize again, obtains immobilized lipase.The standby immobilized lipase of this patent system has good catalysis activity and stability in use, and its catalysis methyl ester conversion rate can reach more than 97%, and recycling batch is 200 times.Publication number is the processing method that the Chinese patent literature of CN101575594A discloses a kind of immobilized candida antarctica lipase B, comprising: the 1) pre-treatment of resin; 2) preparation of immobilized enzyme: pretreated resin is joined in reactor, add again organic medium, after standing, add enzyme liquid, add-on is mass ratio---candida antarctica lipase B: resin=1: after 60～100 sealings, be placed in water bath with thermostatic control shaking table, 1～3h under 25～35 ℃ of conditions, then vacuum lyophilization and get final product.This patent be take organic solvent as mounting medium, realizes the method for lipase immobilization in micro-water, and loss of enzyme activity is less, and the enzyme rate of recovery alive reaches 65.4～83.3%.
As above-mentioned patent, the research major part of at present general lipase immobilization is to improve its recycling number of times, strengthens its reaction stability and reduces the loss that enzyme is lived, lipase activity loses little or is slightly improved, and in immobilization process, still need to use some organic solvents (CN101575594A), activator (CN101280297A), protein-crosslinking agent (CN102839166A) etc., complex steps, cost are high.And in foodstuffs industry, reduce as far as possible the use of organic solvent to guarantee that the security of food applications is the problem that must consider.
With resin, carrying out in the research of lipase immobilization at present, mostly the characteristic of selected resin is not being carried out the description of refinement, also do not exploring immobilization process to Sn-1, the narrow spectrum impact of 3 specific lipase.As specific lipase, in fixation procedure, not only to consider the problem of enzymic activity, how to guarantee that its regiospecificity is also the problem that must consider.
Summary of the invention
The invention provides a kind of immobilization Sn-1, the preparation method of 3 specific lipases, the immobilized enzyme specificity making is good, and enzyme activity is high, and immobilization process is not with an organic solvent, in food, can use safely.
An immobilization Sn-1, the preparation method of 3 specific lipases, comprising:
(1), by Sn-1, it is in 9.0～10.0 damping fluid that 3 specific lipases are dissolved in pH, obtains enzyme liquid;
(2) get resin, pre-treatment is placed in described enzyme liquid adsorbs, and has adsorbed rear washing and has been dried, and makes immobilization Sn-1,3 specific lipases;
Described resin is styrene tyle macroporous adsorption resin, and specific surface area is 200～600m
2/ g, mean pore size is
The application adopts absorption method immobilization Sn-1,3 specific lipases, and utilizing specific surface area is 200～600m
2/ g, mean pore size are
styrene tyle macroporous adsorption resin adsorb, can adsorb appropriate zymoprotein, zymoprotein molecule is fixed on resin equably, and zymoprotein molecule is in best conformation, thereby guaranteed the specificity of enzyme, and greatly improved than enzyme and lived.Preferably, the specific surface area of resin is 450～600m
2/ g, mean pore size is
preferred, the specific surface area of resin is 480～520m
2/ g, mean pore size is
Resin, before absorption, need first carry out pre-treatment, and activation treatment, can adopt conventional means, as carried out according to resin operation instruction.Described Sn-1,3 specific lipases are regiospecificity lipase, derive from microorganism, described microorganism can be aspergillus niger, geotrichum candidum, aspergillus oryzae etc.Preferred, can to select aspergillus oryzae to originate Sn-1,3 specific lipases.
As Sn-1, the dispersion agent of 3 specific lipases, damping fluid maintains suitable pH can make on enzyme liquid, resin zymoprotein molecule in best conformation, thereby guarantee the specificity of immobilized lipase, preferably, the pH of described damping fluid is 9.4～9.6, and described damping fluid is specifically as follows glycine-sodium hydrate buffer solution.
In described glycine-sodium hydrate buffer solution, the concentration of glycine is 0.05～0.1mol/L.When damping fluid ionic strength is relatively low, the avtive spot of lipase molecule is easy to come out, and can improve the enzyme activity of immobilized lipase.Preferably, the concentration of glycine is 0.1mol/L.
Specificity and the enzyme that to enzyme amount, also can affect immobilized lipase are lived, and in step (2), the mass ratio of enzyme and resin is 0.1:1～0.2:1, is preferably 0.10:1～0.15:1, more preferably 0.15:1.Should be under enzyme amount condition, the immobilized lipase enzyme of preparation is lived high, and good stability, improves more than 10 times compared with resolvase is the highest than enzyme work.
Adsorption time is little on the specificity impact of enzyme, but can affect the adsorptive capacity of enzyme, thus the enzyme that affect immobilized lipase live, alive for guaranteeing the enzyme of immobilized lipase, the time of described absorption is 1～3 hour, the temperature of described absorption is generally 25～35 ℃.
In addition, in the immobilization process of lipase, for resin is fully contacted with enzyme, absorption can be carried out under oscillating condition.
After having adsorbed, conventionally need to wash resin, to remove the not lipase of absorption, the solution of washing use can be glycine-sodium hydroxide buffer solution.
The immobilization Sn-1 that the present invention also provides described preparation method to prepare, 3 specific lipases.The immobilized lipase enzyme specificity that the present invention obtains is good, higher than vigor, for the synthetic Reaction time shorten greatly of OPO.
The present invention also provides described immobilization Sn-1, the application of 3 specific lipases in synthetic OPO.
Described application specifically comprises: take plam oil and oleic acid as substrate, with described immobilization Sn-1,3 specific lipases are that catalyzer reacts, and from reactant, separation and purification obtains OPO.
In the process of synthetic OPO, reaction can be carried out in shaking bath or reactor, and shaking table or rotating speed of agitator are generally 150～250r/min.If the too low reaction times consumed of rotating speed is longer, larger to the physical abuse of immobilized lipase enzyme carrier while reacting if rotating speed is too high, be unfavorable for the recycling of immobilized lipase.
Described plam oil can be selected the plam oil (fusing point is 58 ℃) of plant origin, and source is wide, safe.Described oleic acid also can be selected the oleic acid of plant origin, and its purity is preferably in more than 75%.
Described plam oil and the mol ratio of oleic acid are preferably 1:4～1:10.In product, OPO content increases along with the increase of oleic acid consumption, but when adding too much oleic acid, wherein non-oleic acid becomes the carrying out of branch's inhibited reaction, causes the waste of the energy and resource simultaneously.
Described Sn-1, the addition of 3 specificity immobilized lipases is generally 6%～10% (accounting for the amount of reaction substrate), is preferably 8%.
The temperature of described reaction is 50～70 ℃, this temperature is in the suitable temperature of reaction of prepared immobilized lipase, and palmitic fusing point used is 58 ℃ of left and right, after mixing with oleic acid, fusing point decreases, if the too low plam oil of temperature of reaction is difficult to be melted into liquid state, the carrying out that is unfavorable for reaction, temperature of reaction is too high, can accelerate the inactivation of enzyme.Preferably, the temperature of described reaction is 50 ℃
Different enzyme concentrations and substrate mol ratio all can affect the time of reaction.The concrete reaction times can determine according to concrete reaction parameter and concrete product index.General, the described reaction times is 1～4h.
Described separation and purification from reactant obtains OPO, after reaction finishes, from reaction system, isolates immobilized lipase, then removes the free fatty acids in reaction product, and purifying obtains the product that OPO content is higher.Remove free fatty acids and can adopt acid-base neutralisation method or molecular distillation method.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention adopts absorption method, and utilize suitable macroporous adsorbent resin to Sn-1,3 being fixed of specific lipase, the enzyme of the immobilized lipase the making contrast free-fat enzyme be significantly increased (the highest raising more than 10 times) of living, and Sn-1, it is stable that 3 specificitys keep, and specificity quite or is slightly improved compared with resolvase.In addition, the present invention is also clear and definite optimize to reaction conditionss such as enzyme amount, pH, to guarantee that specificity and the enzyme of immobilized enzyme live.
(2) fixing condition of the present invention is gentle, method is simple, use selected resin and enzyme liquid to adsorb under certain condition, in enzyme immobilization process not with an organic solvent, to ensure that immobilized enzyme is in the particularly security in the preparation of infant or baby food raw material of food, and raw material sources are wide, cheap and easy to get.
(3) the immobilization Sn-1 that prepared by the present invention, 3 specific lipases can be used for catalysis plam oil and react generation OPO with oleic acid or oleic acid ester, (immobilized lipase enzyme reaction 1h of the present invention just can make in product indices up to standard to Reaction time shorten greatly, use commercially available immobilized lipase RMIM to need 6h), energy consumption is low, in the reaction product generating, OPO purity is higher, PPP content is lower, meet relevant GB regulation, can be used for allocating baby formula milk powder as food enrichment.
Accompanying drawing explanation
Fig. 1 is each substances content in the middle differential responses time response system of embodiment 27 (immobilized lipase RM IM).
Fig. 2 is each substances content in the middle differential responses time response system of embodiment 28 (immobilized lipase prepared by the present invention).
Embodiment
Except special instruction, the technique means of using in the present invention is the method for well known to a person skilled in the art, following embodiment object is in order to understand better the present invention, but not limits the scope of the invention, and the spirit and scope of the invention are limited by claims.To those skilled in the art, do not deviating under essence of the present invention and scope prerequisite, the various changes that the reaction conditions in these embodiments, separation and Extraction condition are carried out or change also belong to protection scope of the present invention.
Resin calculates as follows to the fixedly rate of lipase:
Fixedly rate=(enzyme liquid protein concentration after the protein concentration-absorption of absorption preferment liquid)/absorption preferment liquid protein concentration * 100%
The enzyme activity of immobilized lipase is used than enzyme work and is represented.The mensuration of living than enzyme take mol ratio that temperature of reaction is 50 ℃, plam oil and oleic acid as 1:8, enzyme concentration as 8%, mixing speed enzyme work of every milligram of lipase albumen under 200r/min condition calculates.And enzyme work is decided by the umol increment of OPO in the unit time.
The Sn-1 of immobilized lipase, 3 specificitys represent with the excessive percentage ratio of class regional isomer (Analogy Regioiso-meric Excess, ARE):
ARE＝(△Sn-1,3-olein％―△Sn-2-olein％)/(△Sn-1,3-olein％+△Sn-2-olein％)
According to < < food safety national standard foodstuff additive 1, in 3-bis-oleic acid-2-palmitin > >, stipulate: 1,3-bis-oleic acid-2-palmitin (OPO) content is in C52 triglyceride level, and tripalmitin in product (PPP) content be also one of important indicator of product (OPO >=40% in product, PPP<10%).
According to < < food safety national standard foodstuff additive 1, in 3-bis-oleic acid-2-palmitin > >, stipulate: 2 palmitinic acids account for the massfraction W of all palmitinic acids
1with %, represent (W
1>=52%), calculation formula is:
W
1＝W
P2/(3*W
P)*100；
W in formula
p2represent 2 palmitic acid contents (%), W
prepresent all palmitic acid contents of sample (%), 3 is reduction factor.
The detection method of lipid acid is with reference to the gas chromatographic analysis > > of GB/T17377-2008 < < animal-plant oil fatty acid methyl ester.
Embodiment 1-7 different resins immobilization Sn-1,3 specific lipases synthetic OPO
With seven kinds of macroporous adsorbent resin DM11, DM130 of vinylbenzene skeleton, AB-8, D3520, NKA, SD300, SD600 fixing Sn-1 respectively, 3 specific lipases, prepare immobilized lipase.The concrete property of seven kinds of resins is in Table 1.
The preparation method of immobilized lipase is:
(1) by 3g lipase powder (Sn-1,3 specific lipases) be dissolved in lixiviate 1h in glycine-sodium hydroxide buffer solution of 100mL pH=9.5,0.1mol/L, the centrifugal 10min of 2000r/min under normal temperature, gets supernatant liquor (being enzyme liquid) for immobilization.
(2) the pretreated resin of 4g is added in 20mL enzyme liquid, vibration absorption 2h in 30 ℃, 200r/min shaking bath, washing suction filtration, lyophilize obtains immobilized lipase (being immobilization Sn-1,3 specific lipases).
The pretreatment process of macroporous adsorbent resin is: resin is immersed in to 1h in 10%NaCl solution, washing suction filtration; In 95% ethanol, soak 1h again, washing suction filtration; In 5%HCl solution, soak 1h again, washing suction filtration is to neutral; In 2%NaOH solution, soak 1h again, washing suction filtration is to neutral.
The immobilized lipase enzymic synthesis OPO that utilizes preparation, method is:
Get 4g plam oil and 11g oleic acid adds 50mL round-bottomed flask, the certain hour that vibrates in 50 ℃, 200r/min shaking bath, question response substrate mixes and when liquid state, adds 1.2g immobilized lipase, reaction 1h; Isolate after immobilized lipase, reaction mixture n-hexane dissolution, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.
The zymologic property that records each substances content and immobilized lipase in reaction product with GC (fid detector) is in Table 1.
Seven kinds of resin immobilization Sn-1 of table 1., 3 specific lipases synthetic OPO
(note: under the same terms, the ratio enzyme of free lipase is lived as 0.104U/mg, specificity ARE=90%.)
As table 1, in seven kinds of macroporous adsorbent resins, the effect of DM11, DM130, AB-8, D3520, NKA (D3520 immobilization effect is best) will significantly be better than SD300 and SD600.This is because the specific surface area of SD300 and SD600 resin is excessive, at 1000m
2/ g left and right, and its mean pore size is less, is only
and zymoprotein molecule is very large, make under this experiment condition, the zymoprotein of its surface adsorption is less, thereby make zymoprotein molecule on its surface undue " unfolding ", cause zymoprotein molecular conformation to change towards unfavorable direction, thereby the ratio enzyme of the enzyme that obtains of immobilization lives lowlyer, and specificity declines to some extent, and the product indices of synthesized is below standard.And the specific surface area of DM11, DM130, AB-8, D3520, five kinds of resins of NKA is all at 200-600m
2between/g, mean pore size exists
between, under this experiment condition, resin surface institute adsorptive enzyme protein content is moderate, zymoprotein molecule is in best conformation, thereby it improves a lot than enzyme specific ionization lipase alive, specificity keeps stable or slightly improves, and the product indices of synthesized can be up to standard, and the reaction times shorter (only 1h).
Embodiment 8-15 is different, and pH damping fluid is prepared immobilization Sn-1,3 specific lipases synthetic OPO
The pH that changes glycine-sodium hydroxide buffer solution, prepares immobilized lipase.
The preparation method of immobilized lipase is:
(1) 3g lipase powder is dissolved in to lixiviate 1h in glycine-sodium hydroxide buffer solution of 100mL0.1mol/L, under normal temperature, the centrifugal 10min of 2000r/min, gets supernatant liquor for immobilization.
(2) the pretreated D3520 resin of 4g is added in 20mL enzyme liquid, vibration absorption 2h in 30 ℃, 200r/min shaking bath, washing suction filtration, lyophilize obtains immobilized lipase.
The pretreatment process of D3520 resin is with embodiment 4.
The immobilized lipase enzymic synthesis OPO that utilizes preparation, method is:
Get 4g plam oil and 11g oleic acid adds 50mL round-bottomed flask, the certain hour that vibrates in 50 ℃, 200r/min shaking bath, question response substrate mixes and when liquid state, adds 1.2g immobilized lipase, reaction 1h; Isolate after immobilized lipase, reaction mixture n-hexane dissolution, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.
The zymologic property that records each substances content and immobilized lipase in reaction product with GC (fid detector) is in Table 2.
Immobilization Sn-1 prepared by the different pH of buffer of table 2., 3 specific lipases synthetic OPO
(note: under the same terms, the ratio enzyme of free lipase is lived as 0.104U/mg, specificity ARE=90%.)
As table 2, this tests lipase used is alkaline lipase, when pH of buffer is between 9.0-10.0, in enzyme liquid, zymoprotein molecule is in best conformation, be fixed on zymoprotein molecule on resin also in best conformation, thereby it improves a lot than enzyme specific ionization lipase alive, it is stable that specificity keeps, the product indices of synthesized can be up to standard, and the reaction times is shorter.And when pH of buffer alkalescence is too low or when too high, in enzyme liquid there is irreversible destruction in the conformation of zymoprotein molecule, thereby make to be fixed on zymoprotein molecule on resin in unfavorable conformation, cause the ratio enzyme of the enzyme that immobilization obtains to live lower, and specificity declines to some extent, the product indices of synthesized is difficult to up to standard.
The immobilization Sn-1 that embodiment 16-20 difference is prepared to enzyme amount, 3 specific lipases synthetic OPO
Change, to enzyme amount, is prepared immobilized lipase.
The preparation method of immobilized lipase is:
(1) a certain amount of lipase powder is dissolved in to lixiviate 1h in glycine-sodium hydroxide buffer solution of a certain amount of pH=9.5,0.1mol/L, under normal temperature, the centrifugal 10min of 2000r/min, gets supernatant liquor for immobilization.
(2) the pretreated D3520 resin of 4g is added in the enzyme liquid of 20mL, vibration absorption 2h in 30 ℃, 200r/min shaking bath, washing suction filtration, lyophilize obtains immobilized lipase.
The pretreatment process of D3520 resin is with embodiment 4.
The immobilized lipase enzymic synthesis OPO that utilizes preparation, method is:
Get 4g plam oil and 11g oleic acid adds 50mL round-bottomed flask, the certain hour that vibrates in 50 ℃, 200r/min shaking bath, question response substrate mixes and when liquid state, adds 1.2g immobilized lipase, reaction 1h; Isolate after immobilized lipase, reaction mixture n-hexane dissolution, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.
The zymologic property that records each substances content and immobilized lipase in reaction product with GC (fid detector) is in Table 3.
The immobilization Sn-1 that table 3. difference is prepared to enzyme amount, 3 specific lipases synthetic OPO
(note: under the same terms, the ratio enzyme of free lipase is lived as 0.104U/mg, specificity ARE=90%.)
As table 3, when to enzyme amount hour, the zymoprotein of resin surface absorption is less, thereby make zymoprotein molecule on its surface undue " unfolding ", cause zymoprotein molecular conformation to change towards unfavorable direction, thereby the ratio enzyme of the enzyme that obtains of immobilization lives lowlyer, and specificity declines to some extent, and the product indices of synthesized is below standard.When being 0.10:1～0.15:1 to enzyme amount, resin surface institute adsorptive enzyme protein content is moderate, and zymoprotein molecule is in best conformation, thereby improve a lot than enzyme contrast alive free-fat enzyme, it is stable that specificity keeps, and the product indices of synthesized can be up to standard, and the reaction times is shorter.
When being 0.20:1～0.25:1 to enzyme amount, resin surface has adsorbed too much zymoprotein, and the resistance to mass transfer forming between zymoprotein molecule makes the reduction alive of the ratio enzyme of immobilized lipase.But when synthetic OPO, because it is larger to enzyme amount, so within the reaction times of 1h, in its product, indices still can be up to standard.
Immobilization Sn-1 prepared by the different adsorption times of embodiment 21-26,3 specific lipases synthetic OPO
Change the adsorption time of enzyme liquid and resin, prepare immobilized lipase.
The preparation method of immobilized lipase is:
(1) 3g lipase powder is dissolved in to lixiviate 1h in glycine-sodium hydroxide buffer solution of 100mL pH=9.5,0.1mol/L, under normal temperature, the centrifugal 10min of 2000r/min, gets supernatant liquor for immobilization.
(2) the pretreated D3520 resin of 4g is added in 20mL original enzyme liquid, vibration absorption certain hour in 30 ℃, 200r/min shaking bath, washing suction filtration, lyophilize obtains immobilized lipase.
The pretreatment process of D3520 resin is with embodiment 4.
The immobilized lipase enzymic synthesis OPO that utilizes preparation, method is:
Get 4g plam oil and 11g oleic acid adds 50mL round-bottomed flask, the certain hour that vibrates in 50 ℃, 200r/min shaking bath, question response substrate mixes and when liquid state, adds 1.2g immobilized lipase, reaction 1h; Isolate after immobilized lipase, reaction mixture n-hexane dissolution, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.
The zymologic property that records each substances content and immobilized lipase in reaction product with GC (fid detector) is in Table 4.
Immobilization Sn-1 prepared by the different adsorption times of table 4., 3 specific lipases synthetic OPO
(note: under the same terms, the ratio enzyme of free lipase is lived as 0.104U/mg, specificity ARE=90%.)
As table 4, as adsorption time (0.5h) more in short-term, resin surface does not adsorb the zymoprotein molecule of enough volumes, cause its fixedly rate, than enzyme, live slightly lowly, in product, indices is below standard.When adsorption time is 1h, resin is to the absorption of zymoprotein molecule in enzyme liquid balance, then extends adsorption time, and resin also no longer increases the adsorptive capacity of zymoprotein molecule, therefore it is than enzyme no longer change alives.In addition, adsorption time is little on the specificity impact of enzyme.
Embodiment 27 immobilized lipase RM IM (Novozymes) catalyze and synthesize OPO
Get 140.76g plam oil and 383.52g oleic acid and add in 1L reactor, under 50 ℃, 200r/min condition, stir certain hour, question response substrate mixes and when liquid state, adds 31.46g RM IM, reaction 9h.Since the 4th hour, the reaction mixture n-hexane dissolution that took a morsel every a hour, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.With GC (fid detector), record each substances content in reaction product and see Fig. 1.
Embodiment 28 immobilization Sn-1,3 specific lipases catalyze and synthesize OPO
60g lipase powder is dissolved in to lixiviate 1h in glycine-sodium hydroxide buffer solution of 2000mL pH=9.5,0.1mol/L, under normal temperature, the centrifugal 30min of 3000r/min, gets supernatant liquor for immobilization.
The pretreated D3520 resin of 200g is added in 1000mL original enzyme liquid, vibration absorption 1h in 30 ℃, 200r/min shaking bath, washing suction filtration, lyophilize obtains immobilized lipase.
The pretreatment process of D3520 resin is with embodiment 4.
Get 140.76g plam oil and 383.52g oleic acid and add in 1L reactor, under 50 ℃, 200r/min condition, stir certain hour, question response substrate mixes and when liquid state, adds 31.46g immobilization Sn-1,3 specific lipases, reaction 9h.The reaction mixture n-hexane dissolution that took a morsel every one hour, then remove free fatty acids with KOH-water-alcohol solution, then rotary evaporation is removed the OPO product that normal hexane obtains high-content.With GC (fid detector), record each substances content in reaction product and see Fig. 2.
By Fig. 1 and Fig. 2, known, 50 ℃ of temperature of reaction, substrate mol ratio 1:8 (plam oil: oleic acid), under the reaction conditions of enzyme concentration 6%, catalyze and synthesize OPO, the prepared immobilized lipase enzyme reaction 1h of the present invention just can make in product indices up to standard, and use commercially available immobilized lipase RM IM, need 6h.Illustrate the prepared immobilized lipase enzyme of the present invention live higher, in the reaction of synthetic OPO, Reaction time shorten preferably.And the preparation cost of the immobilized lipase that the present invention is prepared is lower, the industrialization that is suitable for OPO is synthetic in a large number.
Claims (9)
1. an immobilization Sn-1, the preparation method of 3 specific lipases, is characterized in that, comprising:
(1), by Sn-1, it is in 9.0～10.0 damping fluid that 3 specific lipases are dissolved in pH, obtains enzyme liquid;
(2) get resin, pre-treatment is placed in described enzyme liquid adsorbs, and has adsorbed rear washing and has been dried, and makes immobilization Sn-1,3 specific lipases;
Described resin is styrene tyle macroporous adsorption resin, and specific surface area is 200～600m
2/ g, mean pore size is
2. preparation method as claimed in claim 1, is characterized in that, described Sn-1, and 3 specific lipases derive from aspergillus oryzae.
3. preparation method as claimed in claim 1, is characterized in that, the specific surface area of described resin is 450～600m
2/ g, mean pore size is
4. preparation method as claimed in claim 1, is characterized in that, described damping fluid is glycine-sodium hydrate buffer solution.
5. preparation method as claimed in claim 4, is characterized in that, in described glycine-sodium hydrate buffer solution, the concentration of glycine is 0.05～0.1mol/L.
6. preparation method as claimed in claim 1, is characterized in that, in step (2), the mass ratio of enzyme and resin is 0.1:1～0.2:1.
7. preparation method as claimed in claim 1, is characterized in that, the temperature of described absorption is 25～35 ℃, and the time is 1～3h.
8. the immobilization Sn-1 preparing as claim 1～7 any one preparation method, 3 specific lipases.
9. immobilization Sn-1 as claimed in claim 8, the application of 3 specific lipases in synthetic OPO.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828756A (en) * | 2017-10-12 | 2018-03-23 | 广东惠尔泰生物科技有限公司 | A kind of preparation method of the selectivity immobilized lipases of Sn 1,3 |
| CN108486094A (en) * | 2018-04-20 | 2018-09-04 | 中国科学院南海海洋研究所 | A kind of immobilized lipase and preparation method thereof |
| CN113073090A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Method for immobilizing lipase for enriching polyunsaturated fatty acids |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995339A (en) * | 2006-12-28 | 2007-07-11 | 浙江大学 | Penicillin acylation enzyme-fixing carrier preparation method and carrying method |
| CN102757988A (en) * | 2012-07-25 | 2012-10-31 | 浙江大学 | Preparation method of 1,3-dioleoyl-2-palmitoyl triglyceride |
| CN103805617A (en) * | 2012-11-09 | 2014-05-21 | 丰益(上海)生物技术研发中心有限公司 | 1,3-specific lipase and encoding gene sequence and application thereof |
-
2014
- 2014-07-04 CN CN201410321112.7A patent/CN104140961A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995339A (en) * | 2006-12-28 | 2007-07-11 | 浙江大学 | Penicillin acylation enzyme-fixing carrier preparation method and carrying method |
| CN102757988A (en) * | 2012-07-25 | 2012-10-31 | 浙江大学 | Preparation method of 1,3-dioleoyl-2-palmitoyl triglyceride |
| CN103805617A (en) * | 2012-11-09 | 2014-05-21 | 丰益(上海)生物技术研发中心有限公司 | 1,3-specific lipase and encoding gene sequence and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XUEBING ZHAO ET AL.: "Kinetics of lipase recovery from the aqueous phase of biodiesel production by macroporous resin adsorption and reuse of the adsorbed lipase for biodiesel preparation", 《ENZYME AND MICROBIAL TECHNOLOGY》, vol. 52, 31 December 2013 (2013-12-31) * |
| 刘汝宽 等: "树脂固定脂肪酶技术的研究", 《河南工业大学学报(自然科学版)》, vol. 28, no. 3, 30 June 2007 (2007-06-30) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828756A (en) * | 2017-10-12 | 2018-03-23 | 广东惠尔泰生物科技有限公司 | A kind of preparation method of the selectivity immobilized lipases of Sn 1,3 |
| CN107828756B (en) * | 2017-10-12 | 2020-11-27 | 广东惠尔泰生物科技有限公司 | Preparation method of Sn-1,3 specific immobilized lipase |
| CN108486094A (en) * | 2018-04-20 | 2018-09-04 | 中国科学院南海海洋研究所 | A kind of immobilized lipase and preparation method thereof |
| CN113073090A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Method for immobilizing lipase for enriching polyunsaturated fatty acids |
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