Synthesis of hydrophobic esters with yeast surface displayed-lipase

Esters are commodity chemicals with a wide variety of industrial applications. Traditional methods of synthesis (acid or basic catalysis) require corrosive chemicals, harsh operating conditions, and expensive downstream cleanup. A biocatalytic route (typically employing a lipase) offers an alternative: environmentally friendly catalysts, relatively mild operating conditions, and simpler downstream purification. Lipases are ambassadors for industrial biocatalysis because they are chemically promiscuous, catalyzing hydrolytic and synthetic reactions. The cost of many lipase preparations is prohibitive, however, due in large part to the cost of manufacturing a pure, immobilized (and therefore reusable) form. Yeast surface display (YSD) is a potential platform for enzyme immobilization that combines production, purification, and immobilization, thereby reducing catalyst costs.

We immobilized two lipases via YSD—CalB (Candida antarctica lipase B, Novozym 435) and ycM37L (yeast codon-optimized lipase from Photobacterium lipolyticum sp. M37). The catalyst is prepared by transforming S. cerevisiae with a single-copy, inducible vector that attaches the protein of interest to the outer cell wall by means of an N-terminal fusion to alpha-agglutinin. The yeast are grown on glucose in shake flasks, then used to seed a 2 L bioreactor for induction on galactose. The cells are fixed and dried by lyophilization. The YSD biocatalyst was characterized with hydrolytic activity assays and flow cytometry. The YSD biocatalyst synthesized esters of water-insoluble alcohols and fatty acids in heptane. On a mass loading basis, commercial lipase (Novozym 435) out-performs the YSD biocatalyst. However, estimates of the protein loading reveal a much higher specific activity in the YSD system.