Front Matter

in subcloning the lipase genes into expression plasmids, especially those that had

high copy numbers, extra precautions were taken to ensure tight regulation of ex-

pression. DNAs for the pro- and mature lipase were cloned into a tightly regulated

expression system employingE. coliBL21 (DE3) as host and the plasmid pET11-d

as vector. This cloning system (Studier et al., 1990) contains dual levels of control of

gene expression. Cloned DNAs are placed under the regulatory control of the bac-

teriophage T7 gene 10 promoter. This promoter is recognized by T7 RNA polymer-

ase synthesized by the bacterial host. Synthesis of this polymerase is itself also regu-

lated, and is under the control of alacpromoter. In addition, the cloned lipase gene is

under control of thelacrepressor/operator system. Only by employing this tightly

regulated system was it possible to obtain stable cell lines that grew well in the

absence of inducer, and also synthesized high levels of lipase or prolipase upon

induction. Using these strains, prolipase levels of 9–15 % of total protein, and ma-

ture lipase levels of 15–21 % of total protein, were reached following induction.

At these high expression levels, the lipases were not soluble in the cytoplasm.

Insoluble particles (inclusion bodies) formed and were recovered by centrifugation

following chemical lysis of the cells. Active enzyme was produced by dissolving the

inclusion bodies in 8 M urea and diluting the solution 20-fold into the redox system 1

mM cystine/5 mM cysteine. The active enzymes produced in this manner were pur-

ified according to the protocol developed for the fungal enzyme. The resulting pure

mature lipase had a specific activity comparable to that of authentic fungal enzyme.

The prolipase also assumed an enzymatically active configuration upon renaturation,

and was purified to yield a preparation with a specific activity comparable to that of

the mature lipase. Therefore, the propeptide fragment does not function as an inhi-

bitor of lipolytic activity prior to maturation. SinceE. coliis generally unable to

glycosylate proteins, the recovery of fully active lipase again indicates that glyco-

sylation is not a prerequisite to enzymatic activity. Using this expression route, it was

possible to increase the yields of pure enzyme more than 100-fold compared to those

obtained usingR. delemaras the enzyme source.

These efforts provided the first supplies of prolipase. The enzyme displayed a

specific activity and optimal pH similar to those of the mature enzyme. In the pre-

sence of substrate, it demonstrated the same thermolability as the mature enzyme,

both being quickly inactivated above 30 8 C. However, in the absence of substrate the

prolipase was markedly thermostable, retaining full activity after exposure to tem-

peratures as high as 70 8 C. In contrast, mature lipase was quickly inactivated above

408 C. Apparently, in the absence of a substrate interface the propeptide delays the

onset of thermal denaturation, or promotes rapid renaturation of the enzyme. Another

difference between the two enzymes was that the prolipase was less toxic to its host

than the mature lipase. Thus, it was possible to directly detect and quantitate lipolytic

activity in bacteria. This greatly facilitated the subsequent directed mutagenesis of

the lipase gene, since it provided a means of readily producing sufficient enzyme for

screening and simple characterization without the necessity of isolating and renatur-

ing inclusion bodies. The availability of prolipase also allowed the first crystalliza-

tion and investigation of its three dimensional structure (Swenson et al., 1994).

4.6 Subcloning and regulated overexpression of the lipase gene 79