Recombinant enzymes. Prepared by: Yergaliyeva R. Biotechnology 17-04 Content 1. 2. 3. 4. Why choose a recombinant enzyme? The lipase/acyltransferase from Candida parapsilosis Recombinant Yeast Exo-B-1,3-glucanase expressed in E.Coli Cells Recombinant Drug Metabolizing Enzymes Introduction - Presently, NEB supplies more than 275 restriction enzymes, over 250 of which are available in recombinant form, as well as numerous recombinant polymerases and modifying enzymes for a wide variety of applications. Why choose a recombinant enzyme? - - - Purity: Once an enzyme system is cloned, choice of expression vector and strain background allows tight control over the production environment. For restriction endonucleases, this eliminates enzymes known to contaminate native preparations. Consistency: the yields obtained for recombinant and overexpressed enzymes are significantly larger than those produced by native strains. Production of larger lots means greater product consistency and less lot-to-lot variation. Affordability: At NEB, the introduction of recombinant enzymes has resulted in lower $/unit charges. - - The possibility to clone, express and purify recombinant enzymes have originated the opportunity to dispose of a virtually infinite array of proteins that could be used in the clinics to treat several inherited and acquired pathological conditions. However, the direct administration of these recombinant proteins faces some intrinsic difficulties, such as degradation by circulating proteases and/or inactivation by the patient immune system. The lipase/acyltransferase from Candida parapsilosis - - Candida parapsilosis has been previously shown to produce a lipase (i.e. able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles. The present work describes the cloning and the overexpression of the gene coding for this enzyme. Two ORFs (CpLIP1 and CpLIP2) were isolated. The deduced 465-amino-acid protein sequences contained the consensus motif (G-XS-X-G) which is conserved among lipolytic enzymes. Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase. - - Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C. albicans (42-61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far. Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein. The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase. Recombinant Yeast Exo-B-1,3-glucanase expressed in E.Coli Cells - - Yeast exo-B-1,3-glucanase gene (EXGI) was expressed in E.Coli and the recombinant enzyme was characterized. The recombinant ExgIp had an apparent molecular mass of 45kDa by SDSPAGE and the enzyme has a broad specificity for B-1,3-link-ages as well as B1,6-linkages, and also for other B-glucosidic linked substrates, such as cellobiose and pNPG. Kinetic analyses indicate that the enzyme prefers small substrates such as laminaribiose, gentiobiose, and pNPG rather than polysaccharide substrates, such as laminaran and pustulan. - With the high concentration of laminaribiose, the enzyme catalyzed transglucosidation forming laminarioligoosaccharides. The enzyme was strongly inhibited with high concentrations of laminaratan. The enzyme has very broad specificity for B-glucosidic linkages and strong transglucosidase activity. The enzyme is able to hydrolyze B-1,3-glucan as well as B-1,6-glucan by an exo-type mode of action, yielding only D-glucose. The enzyme don’t act on schizophyllan, the structure of which is a branched B1,3-glucan with one unit of three glucose residues of the repeat unit being substituted at.0-6 with a single B-D-glucose residue. - Exo-B-1,3-glucanase genes (EXG) in yeast have been detected in Saccharomyces cerevisiae and Candida albicans. The Candida Exo-B-1,3-glucanase has been well characterized since the enzyme protein was secreted into the medium. S.cerevisiae usually doesn’t secrete the Exo-B-1,3-glucanase in the medium. The enzyme must be bound to cell walls. They tried to prepare this enzyme by expression of E.coli using S.cerevisiae Exo-B-1,3-glucanase gene. Recombinant Drug Metabolizing Enzymes - - The Corning® Gentest™ Supersomes™ recombinant drug metabolizing enzymes product line includes more than 40 different human and animal cytochrome P450 isoforms, UDP-glucuronsyl transferases (UGTs), FlavinContaining Monooxygenases (FMOs), monoamine oxidases (MAOs), human aldehyde oxidase (AOs), soluble N-Acetyltransferases (NATs), and carboxylesterases (CESs). These metabolic enzyme products are manufactured using baculovirustransfected insect cells and exhibit very high levels of catalytic activities. Sideby-side comparisons of the catalytic activity with pooled human liver microsomes (HLMs) are available for many enzymes. - - - They checked transglucosidase activity of this enzyme. Fig. shows the effect of transglucosidase on hydrolysis on laminaribiose by recombinant Ex1p. Under low conc., laminaribiose was hydrolyzed completely to glucose High conc. of the substrate caused transglucosidation reaction to form trisaccharide during the first 20 min. References: 1. Information on the website of New England BioLabs 2. Article by Neugnot V, Moulin G, Dubreucq E, Bigey F. “The lipase/acyltransferase from Candida parapsilosis: molecular cloning and characterization of purified recombinant enzymes”, Eur J Biochem. 2002 Mar 3. Information on website corning.com 4. Article by Kanako Suzuki, Tomio Yabe, Y.Maruyama “Recombinant Yeast ExoB-1,3-glucanase expressed in E.Coli Cells”, 2011, Bioscience, Biotechnology and Biochemistry.
