| Alkyl glucosides, one of the most promising candidates for new nonionic surfactants inthe next generation, are widely used in many different industries as their excellent features;they are used, for example, in cosmetics, pharmaceuticals, food and detergents etc. Thetraditional chemical synthesis of alkyl glucosides usually produce products composed ofvarious isomers and byproducts. Therefore, the application is mainly concentrated in thedetergent. The use in the fields that require high isomer purity is very limited, for example,biology and medicine. In the recent decade, much attention has been paid on their enzymaticsynthesis through β-glucosidase-catalyzed transferor condensation reactions because of theirregio-and stereo-selectivity and simple and mild conditions without numerous steps ofprotection and deprotection. Meanwhile, β-glucosidase not only catalyze the synthesis ofoligosaccharides with monosaccharide, but also catalyze monosaccharides, oligosaccharidesor activated glycosyl derivatives reacting with fatty alcohols or aromatic alcohols tosynthesize glycosidic compounds. At present, the main problems limiting the application ofthe β-glucosidase is the high cost of purification of β-glucosidase, the low stability in theorganic solvent system, the low to lerance of organic solvents and substrates, some activitylost in the condition of low water activity and so on. To get β-glucosidase with high stability,efficiency and tolerance by cloning, expression, screening and transformation is currently oneof the hotspots.In this study, we get two β-glucosidase genes from Dalbergiaco chinchinensis Pierre andAspergillus aculeatus NO.F-50by cloning and gene synthesis. And eight kinds ofrecombinant Pichia pastoris, which successfully express the two β-glucosidases, wereconstructed. The two β-glucosidases were serected expressed and displayed on the cell surfaceby using pPICK9K and the cell surface displaying vector based on flocculation protein Flo1pN-terminal flocculation functional domain（FS）,-agglutinin（NS）and Pichia pastoris cellwall protein Gcw21p. We found that when the secreted recombinant Pichia pastoris expressABGL and DC-BGL under shake flask conditions, the maximum level of their β-glucosidaseactivity was24.8U/mL and1.09U/mL after7d by inducing with methanol. Of the6recombinant Pichia pastoris displaying of ABGL and DC-BGL on the cell surface, the display system based on-agglutinin and Gcw21p, which were anchored covalently on cellwall, show higher efficiency in the surface-displaying of ABGL and DC-BGL. And they aresuperior to the surface displaying system based on flocculation protein Flo1p, which wasanchored non-covalently on cell wall.In the preliminary study, we found that the yield of Hexyl β-D-glucoside（HG）, Octylβ-D-glucoside（OG）, Decyl β-D-glucoside（DG）and Dodecyl β-D-glucoside（DDG）catalyzed by DC-BGL whole-cell-catalysts, which were11.9%,6.5%,6.6%and3.2%respectively without any optimization of reaction conditions, were higher than that of ABGLwhole-cell-catalysts. And the yield of OG and DG of the former were2~3folds of the laterone, and that of DDG was nearly10times.All this shows that DC-BGL has a higher capacityfor the synthesis of APG compared with ABGL, especially in the synthesis of long chain alkylglycosides.Meanwhile, with the carbon chain length of the fatty alcohol substrate increased,the yield of alkyl glycoside catalyzed by the two β-glucosidases reduced. And the yield ofalkyl glucoside catalyzed bythe three whole-cell-catalysts displaying the same enzymeshowed little differences. The expression levels and the amount of the enzyme moleculesdisplaying on cell surface were the main difference between them, but the amount of theenzyme molecules displaying on one unit cell was the key factor for the reaction yields underthe same conditions. Therefore, when choosing the expression system, we should choose asystem with high efficiency for displaying enzyme molecules, which would help to improvethe enzyme activity of unit mass of cells, and to improve the yield. |
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