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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.

SUBMITTER: Ohtaki S

PROVIDER: S-EPMC1449009 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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

Ohtaki Shinsaku S Maeda Hiroshi H Takahashi Toru T Yamagata Youhei Y Hasegawa Fumihiko F Gomi Katsuya K Nakajima Tasuku T Abe Keietsu K

Applied and environmental microbiology 20060401 4

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. ory ...[more]

PMID: 16597938

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Project description:Hydrophobins are small secreted amphipathic proteins ubiquitous among filamentous fungi. Hydrophobin RolA produced by Aspergillus oryzae attaches to solid surfaces, recruits polyesterase CutL1, and thus promotes hydrolysis of polyesters. Because the N-terminal region of RolA is involved in the interaction with CutL1, the orientation of RolA on the solid surface is important. However, the kinetic properties of RolA adsorption to solid surfaces with various chemical properties remain unclear, and RolA structures assembled after the attachment to surfaces are unknown. Using a quartz crystal microbalance (QCM), we analyzed the kinetic properties of RolA adsorption to the surfaces of QCM electrodes that had been chemically modified to become hydrophobic or charged. We also observed the assembled RolA structures on the surfaces by atomic force microscopy and performed molecular dynamics (MD) simulations of RolA adsorption to self-assembled monolayer (SAM)-modified surfaces. The RolA-surface interaction was considerably affected by the zeta potential of RolA, which was affected by pH. The interactions of RolA with the surface seemed to be involved in the self-assembly of RolA. Three types of self-assembled structures of RolA were observed: spherical, rod-like, and mesh-like. The kinetics of RolA adsorption and the structures formed depended on the amount of RolA adsorbed, chemical properties of the electrode surface, and the pH of the buffer. Adsorption of RolA to solid surfaces seemed to depend mainly on its hydrophobic interaction with the surfaces; this was supported by MD simulations, which suggested that hydrophobic Cys-Cys loops of RolA attached to all SAM-modified surfaces at all pH values. IMPORTANCE The adsorption kinetics of hydrophobins to solid surfaces and self-assembled structures formed by hydrophobin molecules have been studied mostly independently. In this report, we combined the kinetic analysis of hydrophobin RolA adsorption onto solid surfaces and observation of RolA self-assembly on these surfaces. Since RolA, whose isoelectric point is close to pH 4.0, showed higher affinity to the solid surfaces at pH 4.0 than at pH 7.0 or 10.0, the affinity of RolA to these surfaces depends mainly on hydrophobic interactions. Our combined analyses suggest that not only the adsorbed amount of RolA but also the chemical properties of the solid surfaces and the zeta potential of RolA affect the self-assembled RolA structures formed on these surfaces.

Dataset Information

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

Publications

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

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