Table 1.
Predicted orthologous genes of PEP4 and PRB1 in GB-4(0).
The candidate orthologous genes of proteases A and B were searched in the draft genome of strain GB-4(0) using the FASTA program and the amino acid sequences of Pep4 and Prb1 of S. cerevisiae, respectively.
Fig 1.
Conserved catalytic motifs in PaPRO1p and PaPRO2p.
Conserved catalytic motifs were analyzed by pair-wise alignment with Clustal Omega: (A) Pep4 and PaPRO1p and (B) Prb1 and PaPRO2p.
Fig 2.
PaE production in ΔPaPRO1 and ΔPaPRO2 mutants.
Wild-type and ΔPaPRO1 and ΔPaPRO2 gene-deletion mutants of strain GB-4(0) were cultured for 5 and 13 days. PaE and its degraded fragments in the supernatant were detected with anti-PaE antibody. PC, purified PaE as the positive control; black triangle, full-length PaE; gray triangles, degraded fragments of PaE in the strain GB-4(0).
Fig 3.
Complementation of gene deletion in ΔPaPRO1 and ΔPaPRO2 mutants.
Secreted PaE in mutant strains (ΔPaPRO1 or ΔPaPRO2) bearing the plasmids pEmp-PaPRO1, pEmp-PaPRO2 or pEmp, and cultured for 8 days, were detected by immunoblotting with anti-PaE antibody. PC, purified PaE as a positive control; black triangle, full-length PaE; gray triangles, degraded fragment of PaE in strain GB-4(0).
Fig 4.
Effect of ΔPaPRO2 on degradation and PaE activity during large-scale culture with XG8.
(A) Mutant strain ΔPaPRO2 and its parent strain, XG8, were cultured in a jar fermenter. Extracellularly secreted PaE was analyzed by Coomassie Brilliant Blue stain. Black closed triangle, full-length PaE; open triangles, degradation fragments of PaE in strain GB-4(0). (B) Dry cell weight and activity (U/mL) of PaE in strains XG8 and ΔPaPRO2-XG8 based on the result of 6 measurements. The standard deviation was indicated by error bar. (C) The effect of filtration removing the cells from culture on PaE activity (U/mL) in strains XG8 and ΔPaPRO2-XG8 based on the result of 6 measurements. The standard deviation was indicated by error bar.