Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins.
The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizuslipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0-7.0, whereas rRAL was more stable at pH 7.0-10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C4), whereas rRAL had the highest lipase activity toward tricaprylin (C8).
Niu
Wei-ning
WN
Beijing University of Chemical Technology, Beijing, China.
Li
Zhao-peng
ZP
Tan
Tianwei
T
D000595:Amino Acid Sequence; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000737:chemistry
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D014280:Triglycerides; D008049:Lipase