Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae

Abstract

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45 degrees C, 55 degrees C, and 55 degrees C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

Fig. 1

Plasmid used for construction of overexpression vectors. The modified A. oryzae amyB promoter and amyB terminator are located upstream and downstream of a multicloning site, respectively. The factor Xa recognition site and His-tag are positioned between the multicloning site and the amyB terminator. aurA ^r is an aureobasidin A resistance gene from A. nidulans

Fig. 2

Multiple alignment of the amino acid sequences of AO090012000706, AO090701000220, AO090103000026 (CpI), and three known serine-type carboxypeptidases: S. cerevisiae carboxypeptidase Y (CPY; NP_014026), Aspergillus phoenicis CpdS (P52719), and P. janthinellum carboxypeptidase S1 (CPD-S1; AAB28596). The sequence of CPD-S1 contains no preprosequence. The conserved substrate binding motif is indicated by the line (I), and catalytic motifs harboring the amino acid of the catalytic triad (filled lozenge) are marked II–IV. S₁ binding sites are shown by filled reverse triangles, S₁′ binding sites by filled circles, and completely conserved residues by asterisks

Fig. 3

SDS-PAGE profile using culture media (a) and purified recombinant proteins from transformants as samples (b). The proteins contained in culture media and the purified proteins were separated using a 10% SDS gel and visualized with silver nitrate or Coomassie Brilliant Blue. a Media from the culture of A89CS3ocpA (lane 1), A89CS3ocpB (lane 2), A89CS3cpI (lane 3), and A. nidulans A89 (lane 4). bArrowed bands were excised from the gel for identification by peptide mass fingerprinting. Bands appeared at 30 kDa in lanes 3, 5, and 7 correspond to endoglycosidase H. Lane 1 Molecular weight marker; lanes 2, 4, and 6 purified recombinants OcpA, OcpB, and CpI, respectively; lanes 3, 5, and 7 deglycosylated purified recombinants OcpA, OcpB, and CpI, respectively. Deglycosylated recombinant OcpA was shown as two bands (filled arrowheads)

Fig. 4

MS scan analysis of carboxypeptidase activities of recombinants OcpA, OcpB, and CpI. a MS spectrum of angiotensin I. MS spectra of b recombinant-OcpA-digested peptide, c recombinant-OcpB-digested peptide, and d recombinant-CpI-digested peptide. Of recombinants OcpA, OcpB, and CpI, 2, 20, and 17 ng, respectively, were mixed with 1 nmol of angiotensin I, and then the mixture was incubated at 30°C for 180, 10, and 60 min, respectively. The peak at m/z 1,299 correspond to whole angiotensin I (arrows). The two peaks at m/z 1,184 and 1,047 correspond to Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His (gray arrowheads) and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (black arrowheads), respectively

Fig. 5

Effects of protease inhibitors on the carboxypeptidase activities of recombinants OcpA, OcpB, and CpI. PMSF, E64, MIA, pepstatin A, and EDTA were added to the reaction mixture at final concentrations of 1, 0.1 mM, 3 μg/ml, 1, and 1 mM, respectively. The activities for Z-Glu-Tyr of the enzymes with each inhibitor were measured at pH 3.7 at 30°C. Reaction mixtures containing water or dimethyl sulfoxide instead of each inhibitor were used as reference. Relative activities were quantified as the percentage activity of each reference. Open bar recombinant OcpA; filled bar recombinant OcpB; hatched bar recombinant CpI

Fig. 6

Amino acid sequences of N-terminal regions of carboxypeptidases O1 and O2 and OcpA. The N-terminal sequence of carboxypeptidases O1 and O2 were determined using an amino acid sequencer. That of OcpA was deduced from the nucleic acid sequence of ocpA from A. oryzae RIB40 (DOGAN accession no. AO090012000706). Asterisks show completely conserved amino acid residues

Fig. 7

Semiquantitative RT-PCR analysis of ocpA, ocpB, and cpI in A. oryzae RIB40. mRNA was isolated from approximately 0.1 g (semidry wet) of mycelia cultured for 1 day in CDSN (lanes 1, 5, 9, 13), CDGN (lanes 2, 6, 10, 14), CDGM (lanes 3, 7, 11, 15), or CDGMN (lanes 4, 8, 12, 16) media. Each medium contained different carbon and nitrogen sources. CDSN contained 3% starch and 0.3% NaNO₃, CDGN contained 3% glucose and 0.3% NaNO₃, CDGM contained 3% glucose and 0.3% skim milk, and CDGMN contained 3% glucose, 0.3%NaNO₃, and 0.3% skim milk. PCR for amplification of ocpA, ocpB, and cpI was performed using 32 cycles and that for γ-actin was performed using 26 cycles. The PCR amplification of γ-actin using each cDNA as a template was carried out for quantitative control. Negative-control PCR analysis using each mRNA as a template showed no amplified fragments (data not shown)