4 Cloning, Mutagenesis, and Biochemical
Properties of a Lipase from the Fungus
Rhizopus delemar
Michael J. Haas, David G. Bailey, Wilber Baker, Thomas R. Berka, David J.
Cichowicz, Zygmunt S. Derewenda, Robert R. Genuario, Rolf D. Joerger,
Robert R. Klein, Karen Scott and Deborah J. Woolf
4.1 Introduction
Enzymes are attractive catalysts for the conduct of chemical reactions. Compared to
nonbiochemical catalysts, they can offer advantages of improved or unprecedented
substrate and product specificity, activity under gentle reaction conditions, easy pro-
duct clean-up, reduced use of toxic chemicals, and ready production of the catalyst
from renewable resources. However, the development of enzymes for biotechno-
logical catalysis has been hindered by such disadvantages as scarce supply (which
translates to high cost), instability, relatively low reaction rates, and large gaps in the
areas of basic and applied knowledge of the best ways in which they may be used.
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are enzymes that, in aqueous
systems, hydrolyze the ester bonds of water-insoluble substances. As such, they offer
an alternative to high-pressure/high-temperature methods for lipid hydrolysis. The
fact that in systems with low water activity they can also synthesize ester and related
bonds has made them attractive catalysts in other applications. Furthermore, the
discovery that they remain catalytically active in organic solvents, in which the
high solubility of hydrophobic substrates and the low solubility of water foster ester
synthesis and interchange, has greatly expanded the range of applications of these
now popular catalysts (Rubin and Dennis, 1997a,b; Kazlauskas and Bornscheuer,
1998).
We have conducted a program to characterize and modify an extracellular lipase
produced by the mycelial fungusRhizopus delemar. [Now more correctly known as
Rhizopus oryzae, as many individually named isolates in this genus are now recog-
nized to be the same organism (Schipper, 1984)].Rhizopuslipases are attractive
catalysts for lipid modification because they are members of a group of lipases
that are active only against esters of primary alcohols. Thus, in the hydrolysis
and synthesis of glycerides, enzymes in this group are positionally selective, acting
only at the 1- and 3- locations. Such a specificity is useful because alteration of the
fatty acid occupancy at these sites can change the physical properties of a lipid quite
markedly. For example, enzyme-catalyzed, positionally specific, interesterification
allows the synthesis of high-value cocoa butter analogs from low-value tallow (Ma-
crae, 1983), of novel glycerides possessing readily digested medium-chain fatty
acids at their termini (Huang and Akoh, 1996; Soumanou et al., 1998), and of tri-
glycerides that are optimally suited for infant nutrition because their fatty acid com-
Enzymes in Lipid Modification.Edited by Uwe T. Bornscheuer
Copyright2000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. ISBN: 3-527-30176-3
