Novel hydrophobic surface binding protein, HsbA, produced by Aspergillus oryzae

Abstract

Hydrophobic surface binding protein A (HsbA) is a secreted protein (14.5 kDa) isolated from the culture broth of Aspergillus oryzae RIB40 grown in a medium containing polybutylene succinate-co-adipate (PBSA) as a sole carbon source. We purified HsbA from the culture broth and determined its N-terminal amino acid sequence. We found a DNA sequence encoding a protein whose N terminus matched that of purified HsbA in the A. ozyzae genomic sequence. We cloned the hsbA genomic DNA and cDNA from A. oryzae and constructed a recombinant A. oryzae strain highly expressing hsbA. Orthologues of HsbA were present in animal pathogenic and entomopathogenic fungi. Heterologously synthesized HsbA was purified and biochemically characterized. Although the HsbA amino acid sequence suggests that HsbA may be hydrophilic, HsbA adsorbed to hydrophobic PBSA surfaces in the presence of NaCl or CaCl(2). When HsbA was adsorbed on the hydrophobic PBSA surfaces, it promoted PBSA degradation via the CutL1 polyesterase. CutL1 interacts directly with HsbA attached to the hydrophobic QCM electrode surface. These results suggest that when HsbA is adsorbed onto the PBSA surface, it recruits CutL1, and that when CutL1 is accumulated on the PBSA surface, it stimulates PBSA degradation. We previously reported that when the A. oryzae hydrophobin RolA is bound to PBSA surfaces, it too specifically recruits CutL1. Since HsbA is not a hydrophobin, A. oryzae may use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation.

FIG. 1.

Isolation of the 14.5-kDa protein in eluted fractions from octyl-Cellulofine. The culture supernatant of A. oryzae RIB40 grown in CD-PBSA was applied to an Octyl-Cellulofine column. Proteins were eluted from the column by a linear gradient (160 g/liter to 0 g/liter) of ammonium sulfate. The lower arrow indicates the 14.5-kDa protein that eluted close to 0 gof ammonium sulfate/liter.

FIG. 2.

Expression and localization of HsbA. Western blotting with an anti-HsbA polyclonal antibody was performed with proteins (100 μg) from the supernatant, cell wall, cell membrane, and cytoplasmic fractions (A) and supernatants from broths of cultures grown on CD-glucose, CD-BU, CD-SU or CD-PBSA (B) and wheat bran extract (C), following resolution by SDS-PAGE.

FIG. 3.

Binding and release of HsbA to PBSA pellets. PBSA pellets (0.2 g) and purified HsbA (10 μg) were mixed in the presence of NaCl or CaCl₂. (A) PBSA pellets were removed by centrifugation. Supernatants were resolved by SDS-PAGE, and HsbA was identified. (B) HsbA (10 μg) was adsorbed to PBSA pellets (0.2 g) in the presence of NaCl or CaCl₂. The PBSA pellets were washed three times with washing buffer and then mixed with 0.01% (wt/vol) Tween 80, 0.01% (wt/vol) Triton X-100, or 10 mM EDTA. The PBSA pellets were removed by centrifugation, and the HsbA remaining in the supernatants was precipitated with and then detected by SDS-PAGE.

FIG. 4.

HsbA and CutL1 combine to degrade PBSA. HsbA (final concentration, 100 ng/ml) was adsorbed to PBSA in the presence (▴) or absence (○) of 0.1 M CaCl₂. BSA (final concentration, 100 ng/ml) was adsorbed to PBSA as control (▪) in the presence of 0.1 M CaCl₂. PBSA particles were washed three times with buffer. PBSA particles were resuspended in a degradation buffer containing 5-μg/ml CutL1. The OD₆₃₀ value of the PBSA suspension was measured, and the degradation ratio was calculated. The percent degradation (P_d) was calculated as P_d = (1 − C_a/C_b) × 100 (26), where C_a is the concentration of the PBSA microparticles after degradation and C_b is the concentration of the PBSA microparticles before degradation. The data are the means ± standard deviation for six measurements. Statistical analysis was performed with a Student t test. *, P < 0.05.

FIG. 5.

QCM monitoring of interaction between HsbA and CutL1. (A) Interaction of soluble HsbA (curve 1) or BSA (curve 2) with CutL1 coated on a QCM electrode. (B) Interaction of soluble BSA (curve 3) or CutL1 (curve 4) with HsbA coated on a QCM electrode. For curve 1, HsbA (final concentration, 18 to 90 nM) was injected stepwise into an analysis chamber in which a CutL1-coated electrode was immersed. For curve 2, BSA (final concentration, 18 to 90 nM) was injected stepwise into an analysis chamber in which a CutL1-coated electrode was immersed. For curve 3, BSA (final concentration, 18 to 48 nM) was injected stepwise into an analysis chamber in which an HsbA-coated electrode was immersed. For curve 4, CutL1 (final concentration, 18 to 48 nM) was injected stepwise into an analysis chamber in which an HsbA-coated electrode was immersed.