Title: Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation Kohno M, Enatsu M, Funatsu J, Yoshiizumi M, Kugimiya W Ref: J Biotechnol, 87:203, 2001 : PubMed
Random mutagenesis was used to improve the optimum temperature for Rhizopus niveus lipase (RNL) activity. The lipase gene was mutated using the error-prone PCR technique. One desirable mutant was isolated, and three amino acids were substituted in this mutant (P18H, A36T and E218V). The wild-type and this randomly mutated lipase were both purified and characterized. The specific activity of the mutant lipase was 80% that of the wild-type. The optimum temperature of the mutant lipase was higher by 15 degrees C than that of the wild-type. To confirm which substitution contributed to enhancing the optimum temperature for enzymic activity, two chimeric lipases from the wild-type and randomly mutated gene were constructed: chimeric lipase 1 (CL-1; P18H and A36T) and chimeric lipase 2 (CL-2; E218V). Each of the chimeric enzymes was purified, and the optimum temperature for lipase activity was measured. CL-1 had a similar optimum temperature to that of the wild-type, and CL-2 had a higher temperature like the randomly mutated lipase. The mutational effect is interpreted in terms of a three-dimensional structure for the wild-type lipase.
The crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 A. The structure was refined to an R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical alpha/beta hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an alpha-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface.
Title: Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus Kohno M, Kugimiya W, Hashimoto Y, Morita Y Ref: Biosci Biotechnol Biochem, 58:1007, 1994 : PubMed
The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34,000 (B-chain)]. Lipase II has a molecular weight of 30,000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6.0-6.5 and a temperature optimum of 35 degrees C, while, for Lipase II these values were pH 6.0 and 40 degrees C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.
Title: Preliminary investigation of crystals of lipase I from Rhizopus niveus Kohno M, Kugimiya W, Hashimoto Y, Morita Y Ref: Journal of Molecular Biology, 229:785, 1993 : PubMed
Lipase I from Rhizopus niveus consists of two polypeptide chains bound non-covalently. Lipase I has been crystallized in a form suitable for X-ray diffraction analysis using the hanging drop method of vapour diffusion at 20 degrees C. The crystals grew at pH 6.0 to 7.0 using 14 to 16% polyethylene glycol 8000 as the precipitant. The crystals are tetragonal with space group P4(1) (or P4(3)) and cell dimensions of a = b = 83.7 A, c = 137.9 A. There are two protein molecules in the asymmetric unit. The diffraction pattern extends to at least 2.5 A resolution.
Title: Molecular cloning and structure of the gene for esterase from a thermophilic bacterium, Bacillus stearothermophilus IFO 12550 Kugimiya W, Otani Y, Hashimoto Y Ref: Biosci Biotechnol Biochem, 56:2074, 1992 : PubMed
Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.
Title: Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi Kugimiya W, Otani Y, Hashimoto Y, Takagi Y Ref: Biochemical & Biophysical Research Communications, 141:185, 1986 : PubMed
The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.
