Synthesis Of Alkyl Glucoside Catalyzed By β-Glucosidase-Displaying Pichia Pastoris

Alkyl glucosides are newly nonionic surfactant, which are completely biodegradable,non-toxic and no-skin irritants. So they have been widely applicated in many areas such asdetergent, food and cosmetic industries. Now the chemical synthesis of alkyl glucosides isbroadly researched. Comparing with the chemical method, enzymatic synthesis can be carriedout under milder reaction conditions with a higher regioselectivity and stereoselectivity.Aspergillus aculeatus β-glucosidase (A-BGL) and Thai rosewood β-glucosidase(DC-BGL) play a very important role in bioconversion processes. The free β-glucosidase hasthe drawbacks on purification and recycling. For the efficient use of β-glucosidase in anaqueous–organic biphasic medium, it is reported that the immobilization of β-glucosidase iseffective for increasing the stability and accessibility of enzyme. Microorganisms have beenused to display enzymes on the cell surface by fusion with cell-wall-anchored proteins as animmobilized enzyme. β-glucosidase displaying yeast biocatalyst has attracted more and moreattentions, because it can be used for the synthesis of useful product, such as alkyl glucosides.In this paper, the recombinant A-BGL and DC-BGL respectively displayed on thesurface of Pichia pastoris were constructed and used as a biocatalyst to catalyze theglycosylation of glucose and fatty alcohols. Using the recombinant A-BGL displayed on thesurface of Pichia pastoris as biocatalyst, many influential factors on whole-cell synthesis ofalkyl glucosides were studied, including water content, glucose loadings, enzyme loadings,acetic acid-sodium acetate buffer pH and temperature. The optimal reaction conditions were:5mL total reaction volume,0.1g glucose (1-hexanol),0.3g glucose (1-octanol and1-decanol),0.05g biocatalysts(1-hexanol and1-octanol),0.2g whole-cell biocatalysts(1-decanol),0.5mL (1-hexanol),0.75mL (1-octanol) and1mL (1-decano) aceticacid-sodium acetate buffer pH3.0, the rest put1-hexanol,1-octanol and1-decanol,55°C,200r/min. After72h the maximum hexyl and decyl β-D-glucoside yield could be11.69%and3.58%. The highest yield of octyl β-D-glucoside was6.34%after96h.In the synthesis of alkyl glucosides catalyzed by whole-cell catalyst, reaction conditionssuch as water content, glucose loadings, enzyme loadings, acetic acid-sodium acetate bufferpH and temperature are studied. The optimal reaction conditions were:5mL total reactionvolume,0.2g glucose with the best initial water activity0.75,0.3g biocatalysts with the bestinitial water activity0.75,5mL1-hexanol,50°C,200r/min. After96h the maximum hexylβ-D-glucoside yield could be28.04%;0.3g glucose (1-octanol and1-decanol),0.5g glucose(1-dodecanol),0.1g biocatalysts (1-octanol and1-decanol),0.3g biocatalysts (1-dodecanol), 0.5mL (1-octanol and1-decanol) acetic acid-sodium acetate buffer (pH4.0),1.5mL(1-dodecanol) acetic acid-sodium acetate buffer (pH5.0), the rest put1-octanol,1-decanoland1-dodecanol,55°C,200r/min. After96h the maximum octyl, decyl and dodecylβ-D-glucoside yield could be9.68%,8.23%and1.29%, respectively. The enzyme showedhigher transglucosylation yields. PNPG, methyl β-D-glucoside and butyl β-D-glucoside wereused in reaction with fatty alcohols,1-hexanol,1-octanol,1-decanol and1-dodecanol tosynthesize the respective alkyl glucoside. A maximum of13%yield was obtained for dodecylβ-D-glucoside.