Enzymatic Synthesis Of Galactosyl Glycerides And Evaluation Of Its Anti-Microbial Activity

Glyceroglycolipids are important constituents of chloroplast envelope and cell membrane,which are widely exist in plants,animal tissues and microorganisms.It was shown in several studies that glyceroglycolipids had anti-viral,anti-tumor,anti-inflammatory,immunomodulatory and other biological activity,which were widely used in food,biomedical and osmetics industry.Currently,enzymatic method is the main method for the synthesis of glyceroglycolipid.Compared to traditional chemical synthesis,enzyme-catalyzed synthesis is simple,mild,highly productivity and selectivity.The aim of this study is to synthesize and separate galactosyl glyceride with a series of acyl chain length and investigate lipase-catalysis synthesis process and antimicrobial activity of galactosyl glyceride.The main contents are described as follows:1.Separation and purification of galactosyl glycerides.Vsrious mono-and di-galactosyl glyceride with a series of acyl chain(C10～C18:2)in organic solvent was synthesized in this chapter.The galactosyl glycerol and free fatty acid were used as raw materials and Novozyme435 was used as catalyst.The TLC developing agent of galactosyl glyceride was determined:hexanoyl-galactosylglycerol and stearoyl-galactosylglycerol CH2Cl2:CH3OH(9:1,v/v),lauroyl-galactosylglycerol,palmitoyl-galactosylglycerol and oleoyl-galactosylglycerol CH2Cl2:CH3OH(13:1,v/v),linoleoyl-galactosylglycerol CH2Cl2:CH3OH(10:1,v/v).The separation of products was completed by the silica gel chromatography after balancing the column with chloroform.The chloroform solution containing 0.5%methanol and 0.05%acetic acid was used as the first eluent to elute fatty acids.Then a certain percentage of CH2Cl2:CH3OH:H2O solutions were used as the second eluent to seperate digalactosyl glyceride and monogalactosyl glyceride.Finally,methanol was used as the third eluent to elute galactosyl glycerol.The purified galactosyl glycerides were analysized by HPLC-ELSD and MS,and the mobile phase conditions of different galactosyl glycerides were determined.Purity of all galactosyl glycerides was above 97%.2.Synthesis and characterization of palmitoyl-galactosylglycerol.The effects of reaction solvent,Novozyme435 load,substrate molar ratio(galactosylglycerol to palmitic acid),reaction temperature and reaction time on the yield of palmitoyl-galactosylglycerol were investigated.Based on this result,three factors(lipase load,substrate molar ratio and reaction time)were optimized by response surface method(RSM).The yield of palmitoyl-galactosylglycerol was selected as the response value and the optimal conditions were obtained as follows:acetone as reaction medium,Novozyme435 concentration of 12.93 mg/mL,substrate molar ratio of 1:4.49,at 55 ℃ for 27.60 h.Under these conditions,the palmitoyl-galactosylglycerol yield was reach 90.17%.This result was in agreement with the predicted value(90.18%).Finally,the purified products were characterized by NMR and the monopalmitoyl-galactosylglycerol was identified as a mixture of 1-O-palmitoyl-3-O-β-D-galactopyranosyl-glycerol and 3-0-(6’-O-palmitoyl-β-D-galactopyranosyl)-sn-glycerol,the dipalmitoyl-galactosylglycerol was identified as 1-O-palmitoyl-3-0-(6’-O-palmitoyl-β-D-galactopyranosyl)-sn-glycerol.3.Synthesis and characterization of lauroyl-galactosylglycerol.Results from one-factor-one-a-time experiments indicated that the reaction solvent,Novozyme435 load,substrate molar ratio(galactosylglycerol to lauric acid),molecular sieve content,reaction temperature and reaction time had a significant effect on the lauroyl-galactosylglycerol yield.89.21%of yield was obtained by using response surface method(RSM)under the optimized condition:Novozyme435 conversion of 24 mg/mL,galactosylglycerol to lauric acid molar ratio of 1:11.8,reaction time of 25.24 h when the reaction parameters were set as:acetone 1 mL,molecular sieve content of 58 mg/mL and reaction temperature of 55 ℃.The result was roughly identical to the predicted value(89.68%).The purified products were also characterized by NMR in this chapter and the monolauroyl-galactosylglycerol was identified as a mixture of 1-O-lauroyl-3-O-β-D-galactopyranosyl-glycerol and 3-O-(6’-O-lauroyl-β-D-galactopyranosyl)-sn-glycerol,the dilauroyl-galactosylglycerol was identified as 1-O-lauroyl-3-O-(6’-O-lauroyl-β-D-galactopyranosyl)-sn-glycerol.4.Study on the antimicrobial activity of galactosyl glycerides.All the synthesized galactosyl glycerides(C6～C18:2)and galactosylglycerol were submitted for preliminary evaluation of their in vitro antimicrobial activity against the Gram-positive bacteria,Gram-negative bacteria,mycete and yeast.MIC was evaluated by double dilution method.The results shows that all the compounds has antibacterial activity except galactosylglycerol.No inhibitory activity was exhibited by any compounds against fungus.The acyl chain of galactosyl glycerides palys an important role in the antibacterial activity.Compounds bearing the medium-chain acyl have higher antibacterial activity than others.The antibacterial activity of compounds increased when the unsaturation of acyl chain added.Monolauroyl-galactosylglycerol showed the best inhibitory activity against Gram-negative bacteria(MIC of 78 μg/mL)and Gram-positive bacteria(MIC of 156μg/mL)among the tested galactosyl glycerides.Hexanoyl-galactosylglycerol also showed inhibitory against Gram-negative bacteria(MIC of 625 μg/mL).