CN108531522B

CN108531522B - Method for synthesizing monoglyceride by enzymatic catalysis

Info

Publication number: CN108531522B
Application number: CN201810193360.6A
Authority: CN
Inventors: 王永华; 蓝东明; 袁红; 唐薇; 杨博
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-03-09
Filing date: 2018-03-09
Publication date: 2021-09-21
Anticipated expiration: 2038-03-09
Also published as: CN108531522A

Abstract

The invention belongs to the field of bioengineering, and discloses a method for synthesizing monoglyceride by enzymatic catalysis, which comprises the steps of mixing glycerol and fatty acid according to the molar ratio of 1: 0.1-1: 1, reacting at the temperature of 40-70 ℃ and the stirring speed of 200-600 rpm by taking immobilized monoglyceride lipase as a catalyst, continuously removing moisture generated in the reaction process by using a vacuum pump, and finally obtaining a monoglyceride product by centrifugal separation. The monoglyceride lipase is used as a catalyst to synthesize the unsaturated fatty acid monoglyceride through esterification, other glyceride byproducts are not generated in the reaction process, the separation cost of the monoglyceride product is simplified, and the monoglyceride lipase has the advantages of energy consumption reduction, environmental friendliness, mild reaction conditions, high conversion efficiency, high product purity, less enzyme activity loss and the like, and is suitable for large-scale popularization and application.

Description

Method for synthesizing monoglyceride by enzymatic catalysis

Technical Field

The invention belongs to the field of bioengineering, and relates to a method for catalytically synthesizing monoglyceride from marine monoglyceride lipase.

Background

Monoglyceride is a derivative of oil and fat, is a product of combining one hydroxyl group in glycerol with fatty acid, and is a high-efficiency surfactant. The monoglyceride has the effects of emulsification, foaming, dispersion, defoaming, starch aging resistance and the like, is widely applied to various food fields, and is an emulsifier with the best effect for long-term application in the food field. The consumption of monoglyceride exceeds 50% of the total amount of food emulsifier, and the developed countries in Europe and America apply the monoglyceride to the food industry in the thirties of the last century. To date, there are two main approaches to monoglyceride synthesis: chemical synthesis and enzymatic synthesis. The chemical process for producing monoglyceride mainly comprises two methods of ester exchange and esterification, and raw materials of monoglyceride mainly comprise glycerol and fatty acid. Wherein the ester exchange is also called as oil glycerolysis method and alcoholysis. Besides the alcoholysis of fats and esterification of fatty acid esters of glycerol, the chemical production of monoglycerides includes glycidyl method, epichlorohydrin method, chemical group protection method, etc. The enzymatic production of monoglyceride is only performed in the laboratory research stage at present, and the biggest obstacle for limiting the engineering application is that the production efficiency is reduced and the production cost is greatly increased due to the difficulty in contacting the enzyme with a substrate.

Monoglycerides and diglycerides are a class of polyol-type nonionic surfactants. Monoglyceride, diglyceride and their complex, commonly used as emulsifier, stabilizer, foaming agent, lubricant, antiadherent, plasticizer and antibacterial agent, etc., are used in the industries such as daily use chemicals, food, medicine, textile printing and dyeing, metal processing, etc. At present, the monoglyceride and diglyceride are industrially synthesized mainly by a grease glycerolysis method, generally, inorganic base is used as a catalyst at a high temperature (220 ℃ C. and 250 ℃ C.), the grease and the glycerol are catalyzed to react under the protection of nitrogen, and the temperature needs to be rapidly reduced after the reaction is finished, so that the reduction of the monoglyceride content caused by a reversible reaction is prevented. The product is a mixture of monoglyceride, diglyceride, triglyceride, fatty acid and glycerol, the monoglyceride content is generally 30% -40%, and the diglyceride content is 35% -40%. The method requires high temperature, so the method has the advantages of high energy consumption, deep color and more generated byproducts. Especially for unsaturated grease containing double bonds, the double bonds are easy to oxidize at high temperature, and the quality of glyceride products is seriously influenced. And the method for synthesizing the monoglyceride by using the biological enzyme has the advantages of high efficiency, low energy consumption, environmental friendliness and the like. However, the lipase currently used for monoglyceride synthesis is a triglyceride hydrolase or a diglyceride hydrolase, and the removal of residual diglycerides and triglycerides during the reaction is difficult, and the increase of the separation process leads to an increase in production cost.

Disclosure of Invention

The invention provides a method for synthesizing monoglyceride under catalysis of monoglyceride lipase, which adopts esterification of glycerol and fatty acid, and the method has single product and is beneficial to enriching high-purity monoglyceride products.

The technical scheme adopted by the invention is as follows:

an enzymatic catalytic synthesis method of monoglyceride comprises the steps of mixing glycerol and fatty acid according to a molar ratio of 1: 0.1-1: 1, reacting at the temperature of 40-70 ℃ and the stirring speed of 200-600 rpm by using immobilized monoglyceride lipase as a catalyst, continuously removing moisture generated in the reaction process by using a vacuum pump, and finally obtaining a monoglyceride product through centrifugal separation.

Preferably, the molar ratio of glycerol to fatty acid is 1: 1.

Preferably, the fatty acid is linoleic acid.

Preferably, the catalyst is monoglyceride lipase of marine microbial origin.

Preferably, the DNA sequence of the marine microorganism-derived monoglyceride lipase is shown as SEQ ID NO. 1.

Preferably, the reaction temperature is 65 ℃.

Preferably, the stirring speed is 500 rpm.

Preferably, the marine microorganism-derived monoglyceride hydrolase is produced by expression in Escherichia coli.

Compared with the prior art, the invention has the following beneficial effects:

(1) the monoglyceride lipase is used as a catalyst to synthesize the unsaturated fatty acid monoglyceride through esterification, other glyceride byproducts are not generated in the reaction process, the separation cost of the monoglyceride product is simplified, and the monoglyceride lipase has the advantages of energy consumption reduction and environmental friendliness.

(2) The method has the advantages of mild reaction conditions, high conversion efficiency, high product purity and less enzyme activity loss, and is suitable for large-scale popularization and application.

Drawings

FIG. 1 shows the result of SDS-PAGE electrophoresis of GMGL after Ni column purification; 1 is lysate; 2 is lysate supernatant; 3 is 20mM imidazole eluent; 4 is 500mM imidazole eluent.

Detailed Description

The invention mainly relates to a method for catalytically synthesizing monoglyceride by using monoglyceride lipase GMGL from a marine source. On one hand, a series of mutants are rationally designed to improve the esterification activity of monoglyceride lipase by expressing purified enzyme for catalyzing the esterification of glycerol and fatty acid and analyzing the relationship between structure and function on the basis of crystal structure.

Example 1 expression and purification of monoglyceride lipase GMGL

The DNA sequence of the marine source monoglyceride lipase GMGL is artificially synthesized and is shown as SEQ ID NO. 1. Double restriction sites (EcoRI and BgIII) were used to ligate into the pET-30a (+) vector. The recombinant plasmid is transformed into escherichia coli BL 21(DE3) competent cells, the obtained transformant clone is picked and transferred into an appropriate amount of LB culture medium (kanamycin is added to the final concentration of 50 mu g/mL), the culture is carried out at 37 ℃ until the absorbance at 600nm is 0.8, an inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the final concentration of 0.02%, and the induction is carried out for 18h at 20 ℃. After centrifugation at 4200r/min for 30min, the cells were harvested by centrifugation in buffer: 20mM Tris-HCl (Tris-HCl), pH 8.0, cell: buffer 1 (g): buffer was added at a rate of 10(mL) to resuspend the cells. After the thalli is resuspended, the thalli is cracked by an ultrasonic crushing method, and the crushing procedure is as follows: vibration amplitude 70%, 15 min. The resuspended cells were packed in ice and placed on a tray. After crushing, the mixture is centrifuged at 12000r/min for 40min to remove precipitates and other granular impurities. After the supernatant after centrifugation was bound to Ni-NTA affinity media, the media was washed with a buffer containing 500mM sodium chloride, 100mM phosphate buffer, and 20mM imidazole to remove foreign proteins. Finally, eluting the target protein from the affinity medium by using an eluent containing 500mM sodium chloride, 100mM phosphate buffer solution and 500mM imidazole, collecting peak tip proteins of the eluents with different concentrations, and detecting by using SDS-PAGE electrophoresis. The results of SDS-PAGE are shown in FIG. 1. Concentrating the eluate with 10KD tangential flow ultrafiltration membrane, changing to 20mM Tris-HCl, pH 8.0 buffer solution, quickly freezing with liquid nitrogen, and storing in refrigerator at-80 deg.C.

Example 2 immobilization of monoglyceride lipase GMGL

Firstly, weighing 5g of macroporous resin D101 in a small beaker of 100mL, adding 30mL of ethanol (95%), soaking for 24h, filtering, and repeatedly washing the resin with deionized water until the ethanol is completely removed; continuously adding 30mL of hydrochloric acid (5%) into the resin treated in the last step, soaking for 4h, filtering, and repeatedly washing with deionized water until the pH value of the washing liquid is neutral; adding 30mL of NaOH (2%) into the resin processed in the previous step, soaking for 4h, filtering, and repeatedly washing with clear water until the pH value of the washing liquid is neutral; finally, the resin is soaked in phosphate buffer solution with pH of 6.0, the filtration is carried out at intervals, the pH of the filtrate is measured, new buffer solution is continuously added for soaking, the operation is repeated until the pH of the filtrate is 6.0, and the resin is stored in a refrigerator at 4 ℃ after being filtered to be dry. Mixing enzyme solution of monoglyceride lipase and buffer solution in equal volume, adding resin at a ratio of 30mg/g enzyme to resin, shaking in a combined constant temperature gas bath shaker (30 deg.C, 200rmp) for 2 hr, and collecting supernatant and determining protein content in EP tube. Filtering the immobilized enzyme by adopting a proper filtering mode, washing the immobilized enzyme by using the previous buffer solution until no protein exists in the immobilized enzyme filtrate, draining, and drying the prepared immobilized enzyme at 30 ℃ for 8 hours in vacuum.

EXAMPLE 3 catalytic Synthesis of monoglyceride by monoglyceride Lipase GMGL

Glycerol and linoleic acid were added to a 10mL Erlenmeyer flask in a molar ratio of glycerol to linoleic acid of 1:1, 3:1, 5:1, 7:1, 10:1, 20:1, 40:1, supplemented to 2g with a buffer (100mM Tris-HCl pH 8.0), and 900U of immobilized enzyme GMGL was added to the flask for reaction at 65 ℃ with stirring speed of 500rpm for 72 hours. Continuously removing water generated in the reaction process by using a vacuum pump, and finally obtaining a monoglyceride product through centrifugal separation. The formation of the reaction product was detected by HPLC and the oil sample was sampled for 3, 6, 12, 24, 48, 72h, 50. mu.l or 100. mu.l each time, the results are shown in Table 1.

TABLE 1 influence of substrate molar ratio on GMGL catalyzed esterification of linoleic acid and glycerol

Sequence listing

<110> university of southern China's science

<120> method for synthesizing monoglyceride by enzyme catalysis

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Met Thr Glu Thr Tyr Pro Val Val Lys Gly Ala Glu Pro Phe Phe Phe

1 5 10 15

Glu Gly Asn Asp Ile Gly Ile Leu Val Leu His Gly Phe Thr Gly Ser

20 25 30

Pro Gln Ser Met Arg Pro Leu Gly Glu Ala Tyr His Glu Ala Gly Tyr

35 40 45

Thr Val Cys Gly Pro Arg Leu Lys Gly His Gly Thr His Tyr Glu Asp

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Met Glu Lys Thr Thr Cys Gln Asp Trp Ile Asp Ser Val Glu Ala Gly

65 70 75 80

Tyr Glu Trp Leu Lys Asn Arg Cys Gly Thr Ile Phe Val Thr Gly Leu

85 90 95

Ser Met Gly Gly Thr Leu Thr Leu Tyr Met Ala Glu His His Pro Glu

100 105 110

Ile Cys Gly Ile Ala Pro Ile Asn Ala Ala Ile Asn Met Pro Ala Leu

115 120 125

Ala Gly Ala Leu Ala Gly Val Gly Asp Leu Pro Arg Phe Leu Asp Ala

130 135 140

Ile Gly Ser Asp Ile Lys Lys Pro Gly Val Lys Glu Leu Ala Tyr Glu

145 150 155 160

Lys Thr Pro Ala Ala Ser Ile Arg Gln Ile Val Gln Leu Met Glu Arg

165 170 175

Val Lys Thr Asp Leu His Lys Ile Thr Cys Pro Ala Ile Leu Phe Cys

180 185 190

Ser Asp Glu Asp His Val Val Pro Pro Asp Asn Ala Pro Phe Ile Tyr

195 200 205

Asp His Ile Ala Ser Ala Asp Lys Lys Leu Val Arg Leu Pro Asp Ser

210 215 220

Tyr His Val Ala Thr Leu Asp Asn Asp Arg Gln Lys Ile Ile Asp Thr

225 230 235 240

Ser Leu Ala Phe Phe Lys Lys His Ala Asp Arg

245 250

Claims

1. A method for synthesizing monoglyceride by enzyme catalysis is characterized in that: mixing glycerol and fatty acid at a molar ratio of 1:0.1, using immobilized monoglyceride lipase as catalyst, and heating at 65 deg.C^oC. Reacting at a stirring speed of 500rpm, continuously removing water generated in the reaction process by using a vacuum pump, and finally performing centrifugal separation to obtain a monoglyceride product; the monoglyceride lipase is derived from marine microorganisms, and the amino acid sequence of the monoglyceride lipase is shown as SEQ ID NO. 1; the fatty acidIs linoleic acid.

2. The method of claim 1, wherein: the monoglyceride lipase is prepared by expression of Escherichia coli.