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The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed inE. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

IntroductionChitin, a homopolymer of N-acetyl glucosamine (GlcNAc) residues linked by β-1,4 bonds, is widely distributed in nature as a component of crustacean exoskeleton, insect outer shell, diatoms, fungal cell walls, and squid pens. It is the second most abundant biopolymer next to cellulose, as well as a constant source of renewable raw materials on earth (Tharanathan and Kittur, 2003). Two enzymes catalyzing hydrolysis of the chitin chain to its monomer by synergistic and consecutive action are endochitinases (EC 3.2.1.14), which randomly hydrolyze the β-1,4 glycosidic bonds of chitin, and N-acetylglucosaminidases (chitobiase, EC 3.2.1.30), which preferentially break lower chitooligomers to produce N-acetyl glucosamine (GlcNAc) monomers (Patil et al., 2002).Chitinases play important roles in biological activities such as nutrient intake, morphological change, defense, and attack. Fish and squid can digest chitinous substances as food by using chitinases in the stomach and liver (Matsumiya and Mochizuki, 1996;Matsumiya et al., 1998), whereas insects and shellfish use chitinases to degrade chitinous substances in the exoskeleton during ecdysis (Kramer and Koga, 1986). In plants, chitinases serve to attack fungal pathogens that contain chitinous substances for self-defense (Singh et al., 2007). Chitinases have received considerable attention due to potential applications in the biocontrol of plant pathogenic fungi and insects (Patil et al., 2002) as a target for biopesticides. Chitinases play an important role in the virulence of many bacteria and fungi for insects and fungi by lysing their cell walls. In addition, chitinases inhibit spore germination and germ tube elongation of the phytopathogenic fungi (Mathivanan et al., 1998). Chitinases can be used widely in the production of (GlcNAc)n and GlcNAc, the formation of yeast and fungal spheroplast and protoplast, and the bioconversion of chitin waste to single cell protein for animal feed (Mathivanan et al., 1998).Chitinases are produced by various microorganisms such as bacterial (Singh et al., 2008) and fungal antagonists of other fungal mycoparasistes, nematophagous and entomopathogenic fungi, and others (Rocha-Pino et al., 2011). Because of their wide range of biotechnological applications, chitinases have been purified and characterized.

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