| Sugar fatty acid esters are significant nonionic surfactant which produced from renewable and inexpensive substance. Because they are completely biodegradable under aerobic and anaerobic conditions, non-toxic, no-skin irritants, odourless and tasteless, so widely used as emulsifler in food cosmetic and pharmaceutical industries. Sugar esters can be porduced by enzymatic or chemical methods. However, chemical synthesis of sugar fatty acid esters is generally performed as a high temperature esterification in the persence of alkaline catalyst, which is accompanied by high energe consumption, browning of products and low selectivity toward the various hydroxyl groups in sugars. Comparing with the chemical method,enzymatic synthesis can be performed under milder reaction conditions with a higher regioselectivity.Candida antarctica Lipase B (CALB) has a good potential in industrial application, with a wide range of monosaccaride fatty acid esters often as single regiosomers. The free lipase has the drawbakcs of purification and recycling, while the high cost of commercial immobilized CALB limits its industrial application. Compared with immobilized enzymes, enzyme- displaying yeast whole-cell biocatalyst seems to be an alternative due to its simplicity, high enzymatic activity and cost-effective as well. Lipase-displaying yeast whole-cell biocatalyst has attracted more and more attentions since it can be used for the synthesis of useful product, such as bio-surfactant.In this paper, the recombinant CALB displayed on the surface of Pichia pastoris was constructed and used as a whole-cell biocatalyst to catalyze the esterification of sugers and fatty acids. Glucose and fructose as the acyl acceptors and lauric acid as the acyl donor. The thin layer chromatogaphy (TLC) to qualitatively analyze fatty acid ester was developed. The optimal conditions of TLC were: toluene-ethyl acetate-methanol-water (10:5:4.5:0.2, v/v/v/v) as the mobile phase, coloration by sprayingα-naphthol-sulfuric acid. Products was isolated by silica gel column chromatography at first, using the same condition as TLC, and then by preparative liquid chromatography, sugar fatty acids were identified by Liquid Chromatography-Mass Spectrometry and Nuclear Magnetic Resonance.The influential factors on whole-cell synthesis of glucose laurate monoester were studied, such as solvent composition, percentage of Dmethyl sulfoxide in 2-Methyl-2-butanol (v/v), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: 5 mL total reaction volume, 2-Methyl-2-butanol/Dmethyl sulfoxide (30% Dmethyl sulfoxide, v/v), 0.5 g glucose, 1.0 g lauric acid, 0.5 g whole-cell biocatalysts, the best initial water activity of whole-cell biocatalyst was 0.11, temperature 60°C, 200r/min. The maximum yield could be 48.7% after 72 h.In the synthesis of fructose laurate ester catalyzed by whole-cell catalyst, reaction conditions such as solvent composition, biocatalyst concentration, substrate concentration, water activity, amount of molecular sieves and temperature, were also studied. On the basis of single factor optimization, Response Surface Methodology was used to analyse the interaction of fators: catalyst dosage, substrate concentration and temperature. The highest conversion was obtained in 2-Methyl-2-butanol/Dmethyl sulfoxide, Dmethyl sulfoxide percentage 20% (v/v) in 5 mL total reaction volume, fructose 0.36 g, lauric acid 1.08 g, 0.3 g whole-cell biocatalysts, the best initial water activity of whole-cells biocatalyst was 0.11, temperature 56°C, 200 r/min, and 82.5% of the maximum yield achieved after 72 h.To further examine the potential of the whole-cell biocatalyst for sugar esters synthesis, the operational stability of the whole-cell biocatalyst in organic solvents was investigated. The results indicated that the whole-cell catalyst owned good operation stability, after being reused for 15 batches (24 h/batch), the residual activity remained above 77%, compared with the best batch.
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