Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli.
An Escherichia coli expression system was established to produce recombinant extracellular Pseudozyma (Candida) antarctica lipase B (CALB). With the aim of producing the genuine CALB without additional amino acid residues, the mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3) using the pET system. Inducing gene expression at low temperature (20°C) was crucial for the production of active CALB; higher temperatures caused inclusion body formation. Prolonged induction for 48 h at 20°C allowed for the enzyme to be released into the culture medium, with more than half of the activity detected in the culture supernatant. A catalytically inactive CALB mutant (S105A) protein was similarly released, suggesting that the lipid-hydrolyzing activity of the enzyme was not the reason for the release. The CALB production level was further improved by optimizing the culture medium. Under the optimized conditions, the CALB in the culture supernatant amounted to 550 mg/L. The recombinant CALB was purified from the culture supernatant, yielding 5.67 mg of purified CALB from 50 mL of culture. N-terminal sequencing and ESI-MS analyses showed proper removal of the pelB signal sequence and the correct molecular weight of the protein, respectively, confirming the structural integrity of the recombinant CALB. The kinetic parameters towards p-nitrophenylbutyrate and the enantiomeric selectivity on rac-1-phenylethylacetate of the recombinant CALB were consistent with those of the authentic CALB. This is the first example of E. coli-based extracellular production of a CALB enzyme without extra amino acid residues.
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Ayana
A
Ujiie
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Hideo
H
Nakano
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: iwasaki@agr.nagoya-u.ac.jp.
Yugo
Y
Iwasaki
D000595:Amino Acid Sequence; D000596:Amino Acids; D001483:Base Sequence; D055162:Biocatalysis; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000378:metabolism; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014587:Ustilaginales / Q000201:enzymology / Q000235:genetics
D000596:Amino Acids; D003470:Culture Media; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica
BL21(DE3); Extracellular production; Pseudozyma (Candida) antarctica lipase B; Recombinant Escherichia coli; pelB signal sequence