CN102492736B - Enzymatic synthesizing method of N-acetyl glycerol glutamate - Google Patents
Enzymatic synthesizing method of N-acetyl glycerol glutamate Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 14
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- 230000002255 enzymatic effect Effects 0.000 title abstract 2
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Abstract
The invention discloses an enzymatic synthesizing method of N-acetyl glycerol glutamate. The method comprises steps that: N-acylamino acid and glycerol are adopted as substrates, and are subject to a reaction for 1-96h under a temperature of 30-80 DEG C; when the reaction is finished, an obtained reaction liquid is subject to a post treatment process, such that N-acetyl glycerol glutamate is obtained. The esterification enzyme is immobilized lipase Novozyme 435, immobilized lipase Lipozyme RMIM, immobilized lipase Lipozyme TL IM, papain, lipase FAP15 (Japan Amano Pharmaceutical Co. Ltd), acylase or trypsin. The method provided by the invention is advantaged in mild reaction condition, no organic solvent, economic raw material, high product biocompatibility, good quality, and suitability for large-scale productions.
Description
(1) technical field
The present invention relates to a kind of synthetic method of N-ethanoyl L-glutamic acid glyceryl ester, particularly a kind of enzymic synthesis method of N-ethanoyl L-glutamic acid glyceryl ester.
(2) background technology
Yelkin TTS and lysolecithin are the most frequently used in medicine, luxury food and makeup, most typical emulsifying agents, and it comes from natural, environmental friendliness, and toxicity is low, biological degradability is high, and good with many pharmaceutical cpd consistencies.Its main drawback is that water solubility is low, and originate limited (in rapeseed oil, soybean oil, phospholipids content is only 0.3-2.5%) is expensive.The most tensio-active agents that use at present are chemosynthesis, and are unfavorable to water environmental impact, and biological degradability, biocompatibility are poor.Therefore, efficiently, the development requirement of the tensio-active agent of readily biodegradable, good biocompatibility is day by day urgent.The economy of exploitation take amino acid as raw material, have the Yelkin TTS similarity, have simultaneously the obviously tensio-active agent of water-soluble or antimicrobial vigor, have undoubtedly obvious advantage.
The similar owner of phosphatide tensio-active agent based on amino acid pattern will have linear structure (strand), duplex structure (or Shuangzi) and class glyceride structure (phosphatide stand-in) three classes at present.The exploitation of amino acid type surfactant mainly concentrates on front two classes both at home and abroad at present, connects condensation with natural saturated fatty acid, alcohol, amine and different aminoacids by ester bond, amido linkage and obtains.Each of its polar head and hydrophobic tail, so its HLB value adjustment space is little.And the class glyceride structure can be regarded the analogue of mono-glycerides, two sweet esters and phosphatide as, this class tensio-active agent is connected and composed by the glycerine carbon skeleton by 1 molecular polarity head and 2 molecules or 1 molecule hydrophobic tail,, by changing amino acid (polar head) kind or lipid acid (hydrophobic tail) kind and the number of combination, can regulate easily its HLB value and the solubleness in water, oil.
In prior art relevant for the glycine of hydroxyl, tyrosine and alkalescence arginic chemical the enzyme process report, without the synthetic report of the L-glutamic acid glyceryl ester of dicarboxyl.The present invention selects water solubility is high, source is economic L-glutamic acid as polar head, adopts synthesizing amino acid glyceride in the enzyme process solvent-free system, and the synthetic intermediate that provides of the natural phospholipid analogue based on amino acid starting material is provided.Characteristics of the present invention are not that reaction conditions is gentle, not with an organic solvent, raw material economics, the product biocompatibility is high, quality good, is easy to large-scale production.
(3) summary of the invention
The object of the invention is not to provide a kind of enzymic synthesis method of N-ethanoyl L-glutamic acid glyceryl ester, and the method reaction conditions is gentle, not with an organic solvent, raw material economics, the product biocompatibility is high, quality good, is easy to large-scale production.
The technical solution used in the present invention is:
A kind of enzymic synthesis method of N-ethanoyl L-glutamic acid glyceryl ester, described method is: take N-acylamino acid and glycerine as substrate, under condition of no solvent, under the effect of enzyme, 30～80 ℃, reaction 1～96h, reaction finishes the aftertreatment of afterreaction liquid, obtains described N-ethanoyl L-glutamic acid glyceryl ester; Described enzyme is immobilized lipase Novozyme 435, immobilized lipase Lipozyme RM IM, immobilized lipase Lipozyme TL IM, papoid, lipase FAP15 (Japanese field drugmaker), acyltransferase (Acylase) or trypsinase.
In described substrate, the N-acylamino acid is 1: 5～30 with the amount of substance ratio of glycerine.
Described enzyme is preferably immobilized lipase Novozyme 435 or immobilized lipase Lipozyme RM IM.
The quality consumption of described enzyme counts 0.5～10% with the substrate total mass, and the kind of described enzyme is different, and the vigor scope is also not identical, and the definition standard of enzyme activity is not identical yet.
Described reaction solution post-treating method is: after reaction finishes, the 0.5mol/L sodium hydrogen carbonate solution that adds respectively 2 times of the 5%NaCl solution of 1.5 times of reaction solution volumes and reaction solution volumes, be stirred to without bubble formation, filter, filtrate is cooled to the room temperature organic solvent extraction, collect organic phase, be dried to constant weight, obtain described N-ethanoyl L-glutamic acid glyceryl ester; Described organic solvent is normal hexane, chloroform or ethyl acetate.
further, the enzymic synthesis method of described N-ethanoyl L-glutamic acid glyceryl ester, described method is carried out according to the following steps: N-acylamino acid and glycerine is rear as substrate take the amount of substance ratio as 1: 20～30 mixing, be warming up to 70 ℃, under condition of no solvent, under the effect of immobilized lipase Novozyme 435, in the phosphate buffer solution of pH6.0～9.0, 30～70 ℃, reaction 96h, after reaction finishes, the 0.5mol/L sodium hydrogen carbonate solution that adds respectively 2 times of the 5%NaCl solution of 1.5 times of reaction solution volumes and reaction solution volumes, be stirred to without bubble formation, filter, filtrate is cooled to the room temperature organic solvent extraction, collect organic phase, be dried to constant weight, obtain described N-ethanoyl L-glutamic acid glyceryl ester, the quality consumption of described immobilized lipase Novozyme 435 counts 5～10% with N-acylamino acid and glycerine total mass, described organic solvent is chloroform or ethyl acetate, the quality consumption of described phosphoric acid buffer is 10～50%, preferred 20% of N-acylamino acid and glycerine total mass.
The present invention is solvent-free system, and glycerine not only is reactant but also make solvent, and 50～70 degree viscosity reduce greatly.
Esterification yield of the present invention calculates as follows:
Esterification yield=(total amino acid amount before the amount of amino acid of esterification/reaction) * 100%
After reaction finishes; add 5%NaCl solution in order to improve the polarity of solution in reaction solution; reduce the solubleness of product amino acid glyceryl ester; add simultaneously the 0.5mol/L sodium hydrogen carbonate solution to be used for neutralizing unreacted N-acyl glutamic acid; generate sodium salt, filter, filtrate is used organic solvent extraction; N-acyl glutamic acid sodium salt and glycerine enter in the NaCl aqueous solution, and product N-acyl glutamic acid glyceryl ester changes in organic phase and realizes enrichment.
Compared with prior art; beneficial effect of the present invention is mainly reflected in: the present invention selects water solubility is high, source the is economic N-ethanoyl L-glutamic acid polar portion as emulsifying agent; adopt synthesizing amino acid glyceride in the enzyme process solvent-free system; the synthetic intermediate that provides for the natural phospholipid analogue based on amino acid starting material; reaction conditions of the present invention is gentle; not with an organic solvent, raw material economics, the product biocompatibility is high, quality good, is easy to large-scale production.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
N-ethanoyl L-glutamic acid, N-acetyl-l-glutamine, N-benzoyl arginine (99%) are the brilliant pure reagent company limited in Shanghai.Glycerine, AR, Solution on Chemical Reagents in Shanghai company of traditional Chinese medicines group; Lipozyme RM IM, Lipozyme TL IM, lipase Novozyme 435 is letter zymin company limited of Novi, papoid (Nanning biotechnology company limited), lipase FAP15 (Japanese amano pharmaceutical company), Acylase (Japanese amano pharmaceutical company); Trypsin Chinese Medicine Solution on Chemical Reagents in Shanghai company).
Embodiment 1
0.9458g (5mmol) N-acylamino acid is placed in the interlayer beaker, add 9.2g (100mmol) glycerine, be warming up to 50 ℃, under condition of no solvent, add respectively 0.2029g Esterified Enzyme (immobilized lipase Novozyme 435, immobilized lipase Lipozyme RM IM, immobilized lipase Lipozyme TL IM, papoid, acyltransferase (Acylase) or trypsinase), reaction 24h, after reaction finishes, add respectively 5%NaCl solution 30ml and 0.5mol/L sodium hydrogen carbonate solution 40ml in reaction solution, stir 20～30min extremely without bubble formation, filter, filtrate is used ethyl acetate extraction after being cooled to room temperature (25 ℃), collect organic phase, be dried to constant weight, obtain described N-ethanoyl L-glutamic acid glyceryl ester, product is suitably diluted with moving phase, adopt Waters 1525 efficient liquid phase chromatographic analysis, measure the N-acylamino acid amount of esterification, and calculating esterification yield, the results are shown in Table 1, chromatographic column is Lichrosher 100 RP-18 (5 μ m), and the pillar of Lichro CART 250-4 is analyzed in system, and moving phase is 0.05% acetic acid aqueous solution, flow velocity 0.8mL/min, and 30 ℃ of column temperatures, UV-detector, detect wavelength 215nm.
The comparison of table 1 different sources commercial enzyme to the substrate esterification yield
The esterification yield of different enzyme catalysts catalyzing N-ethanoyl L-glutamic acid, the synthetic N-acyl glutamic acid glyceryl ester of glycerine in solvent-free system is in Table 1.Data show; the esterification activity of immobilized lipase under experiment condition (immobilized lipase Novozyme435, immobilized lipase Lipozyme RM IM, immobilized lipase Lipozyme TL IM) obviously is better than the proteolytic enzyme that dissociates (papoid, acyltransferase (Acylase) or trypsinase); reason may be that the immobilized enzyme thermostability is strong on the one hand; improved simultaneously the contact area of substrate and enzyme due to the existence of carrier, so vigor is higher.In immobilized enzyme, immobilized lipase Novozyme 435 catalysis activities are the highest, this with many documents in the multi-functional and highly active report of relevant immobilized lipase Novozyme435 be consistent.Although immobilized lipase Lipozyme TLIM also shows higher esterification yield, experiment finds that reaction is fully broken after finishing, and this should be that its carrier water absorption and swelling is added due to agitation grinding.In addition, the esterification activity of immobilized lipase Lipozyme RMIM also can be accepted.The vigor performance of papoid of surprisingly entertaining very large hope is barely satisfactory, should be that the easy oxidation inactivation of halfcystine in its active centre causes.
Embodiment 2
0.9458g the N-acyl glutamic acid, 9.2g glycerine, 0.2029g immobilized lipase Novozyme435, under differing temps (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃), in 24 hours reaction times, other experimental implementation, with embodiment 1, the results are shown in Table 2.
The impact of table 2 temperature on Novo 435 catalysis L-glutamic acid, glycerine esterification reaction vigor
Under differing temps, 24 hours productive rates of immobilized lipase Novozyme 435 catalyzing Ns-acyl glutamic acid, glycerine esterification reaction are as shown in table 2.Obviously, temperature is very remarkable on the impact of esterification yield, and temperature is increased to 70 ℃ from 30 ℃, and esterification yield improves more than 3 times.Under high temperature, the catalysis activity of enzyme improves on the one hand, though the solubleness temperature of N-acyl glutamic acid in glycerine raises and increase on the other hand, substrate effective concentration increases, thereby reaction efficiency improves greatly.Temperature rising reaction system reduced viscosity in addition, it is also major reason that mass transfer improves.Usually the optimum temperuture of immobilized lipase Novozyme 435 is at 70-80 ℃, but considers that under high temperature, the loss of catalyzer vigor is fast, selects 70 ℃ as optimum temperuture.
Embodiment 3
0.9458g N-acyl glutamic acid; 9.2g glycerine; the immobilized lipase Novozyme 435 of Different adding amount (the quality consumption counts 0.5,1%, 2%, 3%, 5%, 10% with N-acyl glutamic acid and glycerine total mass); temperature 70 C; 24 hours reaction times; other operations, with embodiment 1, the results are shown in Table 3.
The impact of table 3 enzyme dosage on esterification yield
Enzyme addition test-results shows, end product content increase and not obvious in enzyme dosage 0.5-3% scope, even enzyme dosage increases to 10%, esterification yield also only is increased to 65% from 50%.This may be that the enzyme dosage relative amino acid contents is saturated, and the consumption effect that therefore continues the raising enzyme is also not obvious.Many therefore viscosity is larger for the solvent-free and glycerine consumption of reaction system in addition, and mass-transfer efficiency is not high, although enzyme dosage increases, effectively mass transfer is also the inefficient major reason of esterification.
Embodiment 4
0.9458g N-acyl glutamic acid; the quality of glycerine is added according to the mol ratio (5: 1,10: 1,20: 1,30: 1) of glycerine and N-acyl glutamic acid; temperature 70 C; immobilized lipase Novozyme435 addition is 2% of N-acyl glutamic acid and glycerine total mass; reacted 24 hours; other operations, with embodiment 1, the results are shown in Table 4.
The impact of table 4 glycerine, L-glutamic acid mol ratio
Mechanism that it is generally acknowledged enzyme catalysis amino acid, glycerine esterification is that at first enzyme is combined with acry radical donor-amino acid and is dewatered and form enzyme-aminoacyl mixture; then hydroxyl donor-glycerine is attacked enzyme-aminoacyl mixture; generate ester class product, discharge water molecules, enzyme is free out simultaneously.Wherein second step reaction, moisture and with glycerine competition enzyme-aminoacyl mixture, reply and generate amino acid and cause invalid circulation.Accordingly, improve reaction conversion ratio, at first the consumption of enzyme wants saturated amino acid, and secondly the glycerine consumption is excessive, impels molecular balance to move to the esterification direction on the one hand, reduces simultaneously the abortive response that the moisture competition causes.With respect to the N-acyl glutamic acid, glycerine is more cheap and easy to get in addition, and excess of glycerol can improve the utilization ratio of N-acyl glutamic acid, thereby reduces costs.The impact of different ratios is attempted in research, finds that higher glycerine/amino acid molar ratio is conducive to improve the esterification yield of N-acyl glutamic acid.But glycerine is crossed the insoluble solution of low amino acid, thereby reaction efficiency is low.Glycerine is too high also can cause follow-up cost recovery, and the utilization ratio of simultaneous reactions device is also not high, considers 20～30: the 1st, and suitable.
Embodiment 5
0.9458g the N-acyl glutamic acid, 9.2g glycerine, 0.2029g Novozyme435, under 70 ℃, in 1～96 hour reaction times, other operate with embodiment 1, the results are shown in Table 5.
The productive rate of table 5Novo 435 catalyzing Ns-acyl glutamic acid, glycerine esterification reaction different time
Extend the reaction times, N-acyl glutamic acid esterification yield increases obviously.Reacting 24 hours esterification yields is 48.86%, and time lengthening to 96 hour esterification yield is near 90%, considers that the trace of N-acyl glutamic acid in reaction decomposes (5-8%), therefore think that reaction times of 96 hours can realize the conversion of N-acyl glutamic acid substantially.
Embodiment 6
0.9458g the N-acyl glutamic acid, glycerine 9.2g, temperature 70 C, immobilized lipase Novozyme435 addition is 2% of reactant, and the phosphate buffer solution 2mL of different pH reacted 24 hours, and other operate with embodiment 1, the results are shown in Table 6.
24h esterification yield when table 6 adds the phosphate buffer solution of different pH
Usually in the esterification system, the existence of excess water is disadvantageous to the accumulation product.Yet the necessary especially suitable buffered soln of moisture is to keep immobilized lipase enzyme activity conformation necessary, thereby shows best catalysis activity.During without buffered soln, 24 hours esterification yields are about 40%, and the buffered soln that adds pH6-9 all can significantly improve speed of response near 15-40%.Wherein especially with pH 6.0 buffered soln best results, esterification yield 80% in 24 hours, illustrated that this buffered soln provides better ionic environment for Novozyme 435, therefore showed high vigor.In addition, it is also relevant with its solublization to the N-acyl glutamic acid that buffered soln improves the effect of speed of reaction, and in solution, the effective concentration of N-acyl glutamic acid molecule increases, and its speed of response also can correspondingly improve.
Embodiment 7
Experimental implementation is with embodiment 1, and after reaction finished, reaction solution added respectively normal hexane, chloroform, ethyl acetate extraction, and N-acyl glutamic acid sodium salt and glycerine enter in the NaCl aqueous solution, and product N-acyl glutamic acid glyceryl ester changes in organic phase and realizes enrichment.The different solvents effect of extracting is listed in table 7.
The impact of table 7 different organic solvents on N-acyl glutamic acid glyceryl ester purity
Consider the purity of product and the rate of recovery of product, selecting chloroform is best extraction solvent.
Claims (2)
1. the enzymic synthesis method of a N-ethanoyl L-glutamic acid glyceryl ester, it is characterized in that described method is: take N-ethanoyl L-glutamic acid and glycerine as substrate, under condition of no solvent, under the effect of immobilized lipase Novozyme435,70～80 ℃, reaction 72～96h, reaction is carried out aftertreatment to reaction solution after finishing, and obtains described N-ethanoyl L-glutamic acid glyceryl ester; The mol ratio of described N-ethanoyl L-glutamic acid and glycerine is 1:20～30; The quality consumption of described enzyme counts 0.5～10% with the substrate total mass.
2. the enzymic synthesis method of N-ethanoyl L-glutamic acid glyceryl ester as claimed in claim 1, it is characterized in that described reaction solution post-treating method is: after reaction finishes, the 0.5mol/L sodium hydrogen carbonate solution that adds respectively 2 times of the 5%NaCl solution of 1.5 times of reaction solution volumes and reaction solution volumes, be stirred to without bubble formation, filter, filtrate is cooled to room temperature, use organic solvent extraction, collect organic phase, be dried to constant weight, obtain described N-ethanoyl L-glutamic acid glyceryl ester; Described organic solvent is normal hexane, chloroform or ethyl acetate.
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| CN1441848A (en) * | 2000-07-13 | 2003-09-10 | 日本水产株式会社 | Process for production of glycerides with lipases |
| CN101215587A (en) * | 2008-01-03 | 2008-07-09 | 东华大学 | Solvent-free system biological catalysis fast synthesis method for feruloylated glycerol |
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| CN1441848A (en) * | 2000-07-13 | 2003-09-10 | 日本水产株式会社 | Process for production of glycerides with lipases |
| CN101215587A (en) * | 2008-01-03 | 2008-07-09 | 东华大学 | Solvent-free system biological catalysis fast synthesis method for feruloylated glycerol |
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