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Applied and Environmental Microbiology, April 2006, p. 2407-2413, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2407-2413.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Novel Hydrophobic Surface Binding Protein, HsbA, Produced by Aspergillus oryzae

Shinsaku Ohtaki,1 Hiroshi Maeda,2,3,5 Toru Takahashi,1 Youhei Yamagata,1,3 Fumihiko Hasegawa,3 Katsuya Gomi,3,4 Tasuku Nakajima,1,3 and Keietsu Abe1,3*

Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai 985-8555, Japan,1 Tohoku Technoarch Co., Ltd., Sendai 980-8577, Japan,2 The New Industry Creation Hatchery Center, Tohoku University, Sendai 980-8579, Japan,3 Laboratory of Bioindustrial Genomics, Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University, Sendai 985-8555, Japan,4 Chiba Industrial Technology Research Institute, Chiba 264-0017, Japan5

Received 8 July 2005/ Accepted 19 January 2006


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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 CaCl2. 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.


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INTRODUCTION
 
Various types of petroleum-based synthetic polymers currently are being produced worldwide at the rate of approximately 140 million tons/year. Exceedingly large amounts of these polymers are introduced into the ecosystem as industrial waste products (24). Over the past 3 decades, intensive efforts have been made to prepare environmentally friendly polymers that can be easily degraded by microorganisms. As a result, many types of aliphatic polyesters, including polyhydroxyalkanoates, poly({varepsilon}-caprolactone), and poly(L-lactide), have been developed as biologically degradable polymers (11). Polybutylene succinate-co-adipate (PBSA) is produced by the copolymerization of 1,4-butanediol, succinic acid, and adipic acid (4). PBSA is biologically degradable when present in composts, moist soil, fresh water with activated sludge, and seawater (1, 12). Various microorganisms capable of PBSA degradation have been isolated (27, 30).

Current disposal systems cannot recover monomers or oligoesters from biodegradable plastics because almost all the biodegradable plastics used are incinerated with other garbage or degraded to carbon dioxide and water in composts or soil. To develop a biodegradable plastics recycling system, we selected the filamentous fungus Aspergillus oryzae as the host for expressing proteins involved in plastic degradation. Koji molds, including A. oryzae and Aspergillus sojae, have been used extensively for the preparation of traditional Japanese fermentation products such as sake (rice wine), shoyu (soy sauce), and miso (soybean paste) for >1,000 years; the annual production volume of the products is >1 million tons/year by using large-scale solid-state fermentation systems of the koji molds with plant cereals as their substrates (37). Since koji molds can secrete large amounts of proteins, these fungi also are used as hosts for the production of many heterologous proteins, including enzymes, in fermentation industries (2, 6, 10, 23, 28, 29).

A. oryzae RIB40, whose genome has been sequenced (14, 15), was grown in a minimal medium containing a PBSA emulsion as a sole carbon source, where it produces a polyesterase cutinase and can catabolize PBSA (17, 26). Cutinase, a well known cutin-degrading estrase, is produced by fungal plant pathogens such as Fusarium solani f. sp. pisi and Magnaporthe grisea (22, 25) and degrades the biodegradable poly({varepsilon}-caprolactone plastic (19). The cutinase produced by A. oryzae was designated CutL1 (21) and can degrade polybutylene succinate, PBSA, and poly(L-lactide) (17). When A. oryzae RIB40 is cultivated in a medium containing PBSA as a sole carbon source, the rolA gene encoding the hydrophobin RolA also is highly expressed, and RolA is involved in the degradation of PBSA (26).

Hydrophobins are small proteins containing eight conserved cysteine residues, which are ubiquitous among filamentous fungi (3, 32, 33). Hydrophobins form protective surface coatings for fungi and reduce water surface tension, both of which are needed for growth delimitation of aerial structures such as hyphae and conidiospores (35). Hydrophobins can adsorb to hydrophobic surfaces and to interfaces between hydrophobic (air, oil, and wax) and hydrophilic (water and cell wall) phases (34, 36). The hydrophobin RolA adsorbs to the hydrophobic PBSA surface by recruiting CutL1, resulting in CutL1 accumulation on the PBSA surface and consequent stimulation of PBSA hydrolysis (26). Interaction between the amphipathic protein and the hydrolytic enzyme is a novel mechanism for degradation of a hydrophobic solid material at a solid-liquid interface (26).

Based on the function of the hydrophobin RolA, we hypothesized that proteins in addition to RolA are biologically functional at a solid-liquid interface. The objective of the present study is to explore novel surface-active proteins other than RolA that can contribute to the degradation of hydrophobic materials such as PBSA. If the number of species of such surface-active proteins were expanded, these novel proteins would find great application in the degradation of various types of biodegradable plastics or hydrophobic biomass in industrial solid-state fermentation systems or composters. In the present study, we report on the biochemical properties of a novel hydrophobic surface binding protein A (HsbA) that stimulates PBSA degradation.


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MATERIALS AND METHODS
 
Strains, media, and growth conditions.
Aspergillus oryzae RIB40 (ATCC 42149), which was previously analyzed in A. oryzae expressed sequence tag and genome sequencing projects (14-16), was used as a DNA donor and for protein isolation. Aspergillus oryzae niaD300, an niaD mutant derived from RIB40, was used as the recipient for transformation and protein expression experiments. A strain termed gla-cut is a CutL1-overexpressing strain derived from niaD300 (26). Instead of glucose, 1% (vol/vol) PBSA microparticles (1 µm in diameter; Showa Highpolymer Co., Ltd., Tokyo, Japan) (Czapek Dox [CD]-PBSA), 10-g/liter (wt/vol) succinate (CD-SU), or 10-g/liter 1,4-butanediol (CD-BU) was added to CD liquid medium as the sole carbon source. To obtain purified HsbA and CutL1, an HsbA-overexpressing strain (gla-HsbA) and gla-cut were grown in YPM liquid medium (10-g/liter yeast extract, 20-g/liter polypeptone, and 20-g/liter maltose). Escherichia coli XL1-Blue (Stratagene, Tokyo, Japan) was used for plasmid construction. Escherichia coli was grown at 37°C in LB medium (20-g/liter tryptone, 10-g/liter yeast extract, and 20-g/liter NaCl).

Isolation of HsbA from culture broth of A. oryzae RIB40 grown in CD-PBSA.
The conidiospore suspension of A. oryzae RIB40 was inoculated into 1 liter of CD-PBSA liquid medium to obtain a final concentration of 106 spores/ml. After aerobic cultivation at 30°C for 7 days, the supernatant was obtained by filtration through Miracloth (Calbiochem, Darmstadt, Germany). Ammonium sulfate was added to the supernatant to 160 g/liter, and the mixture was centrifuged (10,000 x g for 40 min at 4°C). The supernatant was loaded onto an Octyl-Cellulofine column type S (3 cm [diameter] by 8 cm; Seikagaku Kogyo Co., Tokyo, Japan) equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 160 g/liter of ammonium sulfate. The column was washed with 1 liter of the same buffer and the proteins with a 160-g/liter to 0-g/liter linear gradient of saturated ammonium sulfate. The eluted fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 17.5% slab gel (13). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane with a Trans-Blot SD semidry transfer cell (Japan Bio-Rad Laboratories, Inc., Tokyo, Japan). The proteins were stained with Coomassie brilliant blue R-250 and excised from the blot. A protein of an ~14.5-kDa molecular mass was eluted from the column close to 0-g/liter saturation of ammonium sulfate. The N-terminal amino acid sequence of this protein was analyzed with an Applied Biosystems 473A protein sequencer (Applied Biosystems, Tokyo, Japan).

Creation of HsbA-overexpressing strain.
An expression plasmid for HsbA (pNG-gla-hsbA) was constructed as follows. DNA fragments were generated by PCR by using primers hsbA-sense (5'-CTTGCATTCAAGTCGACCTGAACAC-3') and hsbA-anti (5'-CTATTGAACTATGCTTCTAGAAGGCCTAATC-3'), and an A. oryzae RIB40 genomic DNA (18) or cDNA library when RIB40 was cultured in CD-PBSA liquid medium (26) as the template (30 cycles, each consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min). The DNA fragments were double digested with SalI and XbaI, and the digested fragment was ligated into the SalI-XbaI site of pUC119 (TaKaRa Bio Co., Shiga, Japan). The insert fragments were sequenced, and cDNA of hsbA gene was obtained. The insert fragment from RIB40 genomic DNA was transferred to the SalI-XbaI site of pNGA142, which contains the maltose-inducible glaA142 promoter (9). This plasmid was designated pNGA-glaA-hsbA. A circular form of pNGA-gla-hsbA was digested with MunI and introduced into A. oryzae niaD300 (niaD) by a protoplast-polyethylene glycol transformation method (18). Transformant homokaryons were purified for three cycles by filtration of conidia through PVDF filters (pore size, 5.0 µm; Millipore) prior to subculture on CD agar plates, as previously described (8).

Five independent homokaryons were selected, and integration of the expression vector at the niaD locus was confirmed by colony PCR with the hsbA sense primer and the niaD primer (26) (5'-GTAGAATCACGAATGAGACCTTTGACGACC-3') and with A. oryzae genomic DNA as the template (30 cycles, each consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min). The conidia of the potential HsbA-overexpressing strains were inoculated into YPM liquid medium (3 ml; 106 spores/ml). After aerobic cultivation for 24 h at 30°C, the culture broth was filtered through Miracloth (Calbiochem). The supernatant (0.4 ml) was precipitated with trichloroacetic acid (TCA) and subjected to SDS-PAGE. The proteins were transferred to a PVDF membrane, and N-terminal sequences of protein bands with a 14.5-kDa molecular mass were determined by the method described above. N-terminal sequences of the 14.5-kDa protein from the five homokaryons corresponded to that of native HsbA. One of the HsbA-overexpressing strains was designated A. oryzae gla-hsbA.

Purification of heterologously produced HsbA and CutL1.
Conidia of gla-hsbA were inoculated into 1 liter of YPM liquid medium (106 spores/ml) and cultivated for 24 h at 30°C. The culture broth was filtered through Miracloth (Calbiochem), and ammonium sulfate was added to the filtrate to 160 g/liter and then centrifuged (10,000 x g; 40 min at 4°C). The supernatant was applied to hydrophobic columns, such as an Octyl-Cellulofine type S (3 cm [diameter] by 18 cm) and a phenyl-Sepharose CL-4B column (3 cm [diameter] by 18 cm) (Amersham Biosciences). The hydrophobic columns were washed and proteins were eluted as described above. The fraction containing HsbA was dialyzed against 10 mM sodium citrate buffer (pH 4.0) and then applied to an S-Sepharose FF column (2 cm [diameter] by 8 cm; Amersham Biosciences, Uppsala, Sweden) equilibrated with the same sodium citrate buffer. Purified HsbA was eluted with a 0 to 0.5 M linear gradient of NaCl. A guinea pig polyclonal antibody against the purified HsbA was prepared commercially (T. K. Craft, Gunma, Japan). CutL1 and anti-CutL1 mouse antibodies were prepared as described by Maeda et al. (17) and Takahashi et al. (26). Protein concentrations were determined with a BCA Protein Assay Reagent kit (Pierce Biotechnology, Inc., Rockford, Ill.).

Localization of HsbA.
Mycelia of A. oryzae RIB40 cultured in CD-PBSA liquid medium were collected with Miracloth (Calbiochem), and the supernatant was prepared as described above. The cell wall protein, membrane proteins, and cytoplasmic proteins were fractionated as previously described (26), and each fraction was resolved on SDS-PAGE. The proteins were transferred to a PVDF membrane, and HsbA was detected by Western blot analysis with an anti-HsbA polyclonal antibody for the primary antibody and alkaline phosphatase-conjugated goat anti-guinea pig immunoglobulin G antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) for the secondary antibody. In immunostaining the subcellular fractions, cutinase CutL1 (17), hydrophobin RolA (26), and mitogen-activated protein kinase HogA (5) were used as markers for extracellular, cell wall, and cytoplasmic fractions, respectively.

Expression of HsbA.
A. oryzae RIB40 was inoculated into 100 ml containing CD-glucose, CD-PBSA, CD-SU, or CD-BU liquid medium to a final concentration of 5 x 106 spores/ml. The spores in CD-glucose were cultured for 2 days at 30°C, and those in the other media were cultured for 5 days at 30°C. The supernatants were prepared as described above. Wheat bran (5 g) was mixed with 4 ml distilled water for 30 min at room temperature and then autoclaved at 120°C for 50 min. The wheat bran medium was inoculated with 106 conidia and incubated at 30°C for 60 h. The wheat bran medium was added to extraction buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 0.01% Tween 80) and stirred at 4°C for 12 h. The mixture was filtered with filter paper (ADVANTEC, Tokyo, Japan). The supernatants and wheat bran extract were resolved by SDS-PAGE and transferred to a PVDF membrane, and HsbA was detected by Western blot analysis as described above.

Binding of HsbA to PBSA pellets.
One milliliter of purified HsbA (10 µg/ml) was dissolved in 10 mM Tris-HCl buffer (pH 8.0) and 0.2 g of PBSA pellets mixed in glass test tubes and shaken gently at 30°C for 90 min. To enhance adsorption of HsbA on the PBSA pellets, NaCl (final concentration, 0.1 to 0.5 M) or CaCl2 (final concentration, 10 µM to 100 mM) was added to the buffer. The PBSA pellets were centrifuged (17,000 x g for 5 min at 4°C), and 0.8 ml of the supernatant was precipitated with TCA and subjected to SDS-PAGE.

Release of HsbA from PBSA.
HsbA solution (50 µg/ml) was adsorbed onto 0.2 g of PBSA pellets by the addition of NaCl (final concentration, 1.0 M) or CaCl2 (final concentration, 0.1 M) to the buffer. The PBSA pellets were washed three times with 10 mM Tris-HCl buffer (pH 8.0). The PBSA pellets were suspended in 1.0 ml of 0.01% (wt/vol) Tween 80, 0.01% (wt/vol) Triton X-100, and 10 mM EDTA and shaken (125 rpm) at 30°C for 90 min. The PBSA pellets were centrifuged (17,000 x g for 5 min at 4°C), and 0.8 ml of the supernatant was precipitated with TCA and subjected to SDS-PAGE.

Degradation of PBSA microparticles with HsbA and CutL1.
PBSA microparticles (0.1% [vol/vol]) suspended in 10 mM Tris-HCl buffer (pH 8.0) were degraded by two methods. First, PBSA microparticles were precoated with purified HsbA (final concentration, 100 ng/ml) or bovine serum albumin (BSA) by the addition of NaCl (final concentration, 1.0 M) or CaCl2 (final concentration, 0.1 M) for 2 h at 30°C with shaking (125 rpm). Then, the PBSA microparticles were washed three times with 10 mM Tris-HCl buffer (pH 8.0). The microparticles were hydrolyzed with purified CutL1 (5-µg/ml final concentration) at 40°C for 120 min. The decrease in turbidity (optical density at 630 nm [OD630]) was measured at 10-min intervals. Next, HsbA and CutL1 were added simultaneously to a suspension of PBSA microparticles, and the mixture was incubated at 40°C for 120 min. PBSA microparticles were diluted and monitored at OD630 to generate a standard curve. The concentration of the PBSA microparticles was determined by fitting the turbidity of the PBSA microparticles to the standard curve. The percent degradation (Pd) was calculated as Pd = (1 – Ca/Cb) x 100 (26), where Ca is the concentration of the PBSA microparticles after degradation and Cb is the concentration of the PBSA microparticles before degradation.

Analysis of the RolA-CutL1 interaction with a QCM.
Quartz crystal microbalance (QCM) measurements were made with a 27-MHz QCM (Affinix Q; Initium Co., Tokyo, Japan). A QCM electrode (Initium Co.) was prepared by immobilization of 10 µl of purified HsbA or CutL1 (250 µg/ml in 10 mM Tris-HCl buffer, pH 8.0) directly onto an electrode. The analysis chamber was filled with 10 ml of running buffer (10 mM Tris-HCl [pH 8.0]; 30°C), and an electrode with immobilized HsbA or CutL1 was immersed in the buffer. A dialyzed CutL1 solution (1.0 mg/ml) was injected stepwise into the analysis chamber, and QCM values were measured as a function of the concentration of CutL1 (10 to 48 nM). A dialyzed HsbA solution (1.3 mg/ml) was injected stepwise into the analysis chamber, and QCM values were measured as a function of the concentration of HsbA (18 to 90 nM). As a negative control, BSA was injected stepwise into the analysis chamber in which the HsbA- or CutL1-coated electrode was immersed. The binding affinity of CutL1 to HsbA was evaluated by frequency changes after CutL1 was injected by plotting a Michaelis-Menten graph (7). The dissociation constant (KD) was calculated from the equation KD = Bmax/2, where Bmax is the maximum binding of analyte to ligand. Coimmunoprecipitation of soluble-state HsbA and CutL1 was performed by the methods of Takahashi et al. (26).


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RESULTS
 
Isolation of HsbA from PBSA degradation product by A. oryzae.
We isolated a 14.5-kDa protein (Fig. 1), following the growth of A. oryzae RIB40 in CD medium with PBSA as the sole carbon source. The N-terminal sequence of this protein was DASAVLADFN, which matched with the A. oryzae genome database, and the cDNA sequence of hsbA gene (National Center for Biotechnology Information [NCBI] accession number AB219422) were cloned. The genomic sequence of hsbA had a 59-bp insertion located 337 bp from the initiation codon. The amino acid sequence of HsbA was 44% identical to Aspergillus fumigatus hypothetical protein Afu2g17630 (NCBI accession number EAL94060), 32% identical to Aspergillus fumigatus antigenic cell wall galactomannoprotein (NCBI accession number EAL84331), 31% identical to Metarhizium anisopliae 4MeS (NCBI accession number EAA58613), and 28% identical to Aspergillus nidulans 6795.2 (NCBI accession number AAB69311). In the A. flavus Gene Index at The Institute for Genomic Research (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=a_flavus), there is an orthologue with 100% identity to the HsbA amino acid sequence. In the Aspergillus oryzae genome databases (14, 15), there was a previously unrecognized orthologue of HsbA-HsbB (27% identity; NCBI accession number AB239469). All of the orthologues except the hypothetical protein Afu2g17630 contain deduced KexB (18) processing sites (Lys-Arg or Arg-Arg) that precede the biochemically determined N-terminal sequence of HsbA.


Figure 1
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  		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.

In vivo expression and localization of HsbA.
Mycelia were disrupted and fractionated into extracellular, cell wall, cell membrane, and cytoplasmic fractions with HsbA detected in the extracellular fraction but not in the cell wall, cell membrane, or cytoplasmic fractions (Fig. 2A). HsbA was expressed only in media with PBSA as the sole carbon source but not if glucose, succinate, or 1,4-butanediol was the sole carbon source (Fig. 2B). HsbA was expressed on solid-state wheat bran medium (Fig. 2C).


Figure 2
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  		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.

Binding and release of HsbA to and from PBSA pellets.
If purified HsbA solution and PBSA pellets (average diameter, 1 mm) were mixed and shaken gently for 90 min at 30°C, the HsbA did not bind to the PBSA pellets, but if NaCl or CaCl2 was added, then HsbA absorbed to PBSA in a concentration-dependent manner (Fig. 3A). The PBSA pellets did not absorb BSA, even in the presence of salts. HsbA was released from the PBSA pellets in the presence of 0.01% Tween 80 or 0.01% Triton X-100 and either NaCl or CaCl2 or 10 mM EDTA and CaCl2 (Fig. 3B).


Figure 3
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  		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.

Degradation of PBSA microparticles by purified CutL1 and HsbA.
When HsbA and CutL1 were concomitantly added to a suspension of PBSA microparticles, HsbA-dependent PBSA degradation via CutL1 was not observed (data not shown). If the PBSA particles were precoated with HsbA and then mixed with CutL1, HsbA clearly stimulated PBSA degradation via CutL1 (Fig. 4). BSA was used as a control of HsbA and did not stimulate PBSA degradation (Fig. 4).


Figure 4
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  		FIG. 4. HsbA and CutL1 combine to degrade PBSA. HsbA (final concentration, 100 ng/ml) was adsorbed to PBSA in the presence ({blacktriangleup}) or absence ({circ}) of 0.1 M CaCl2. BSA (final concentration, 100 ng/ml) was adsorbed to PBSA as control ({blacksquare}) 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 (Pd) was calculated as Pd = (1 – Ca/Cb) x 100 (26), where Ca is the concentration of the PBSA microparticles after degradation and Cb 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.

Interaction of HsbA and CutL1.
Prior to the analysis of interaction of HsbA and CutL1, we confirmed whether HsbA was correctly attached to the surface of a QCM electrode. If HsbA was correctly immobilized on the QCM electrode, anti-HsbA antibodies interacted with HsbA adsorbed on the QCM electrode. When a solution of anti-HsbA polyclonal antibody was injected into the QCM analysis chamber in which an HsbA-coated electrode was immersed, the electrode response was achieved to equilibrium of adsorption of the antibody to HsbA immobilized on the electrode (about 740 Hz). The positive response of the HsbA-precoated electrode indicated that the HsbA-coated electrode could respond to molecules that had specific affinity to HsbA. When the BSA solution was injected into the QCM analysis chamber, the electrode scarcely responded (Fig. 5B, curve 3). If the CutL1 stock solution (1 mg/ml) was injected stepwise into the analysis chamber to a final concentration of 10 to 48 nM (Fig. 5B, curve 4), then specific interaction of CutL1 with the adsorbed HsbA could be detected. The KD was 1.4 x 10–8 M, and the apparent maximum binding (Bmax) of CutL1 to the HsbA adsorbed to the electrode was 1.9 ng/mm2. These results suggest that HsbA (ligand) adsorbed to the QCM electrode had high affinity for soluble CutL1 (analyte).


Figure 5
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  		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.

When anti-CutL1 antibody or anti-HsbA antibody was added to a mixture of CutL1 and soluble-state HsbA in the absence of any hydrophobic materials, coimmunoprecipitation of soluble-state HsbA with CutL1 was negligible (data not shown). These results suggest that HsbA adsorbed to hydrophobic surfaces specifically interacted with CutL1 but that soluble-state HsbA did not.


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DISCUSSION
 
In the present study, we isolated and characterized a novel 14.5-kDa protein HsbA from the culture broth of A. oryzae grown in a medium containing PBSA as the sole carbon source. We demonstrated that HsbA adsorbed on the hydrophobic surface of PBSA specifically recruits CutL1 to the PBSA surface, resulting in CutL1 accumulation on the PBSA surface and consequent stimulation of PBSA hydrolysis.

A. oryzae RIB40 can degrade and utilize the biodegradable plastic PBSA as a sole carbon source (17). When A. oryzae RIB40 catabolizes PBSA, the hydrophobin RolA (26) is highly expressed. RolA promotes CutL1-dependent hydrolysis of PBSA, and when RolA is adsorbed on the hydrophobic surface of PBSA it recruits CutL1, resulting in CutL1 accumulation on the PBSA surface and the hydrolysis of PBSA (26). The discovery of the novel functions of RolA suggests that proteins other than RolA are biologically functional at a solid-liquid interface. We screened proteins that interact hydrophobically with a column of Octyl-Cellulofine from a culture broth of A. oryzae grown in a medium containing PBSA as the sole carbon source and found several surface active proteins (data not shown). The 14.5-kDa protein, HsbA, can bind to PBSA and recruit cutinase CutL1. HsbA does not contain any cysteine residues, so it is not formally classifiable as a hydrophobin (3, 31, 32). Genes orthologous or paralogous to hsbA in other fungi are known (EAL94060, EAL84331, EAA58613, and AAB69331), but none of the orthologues have been functionally characterized. Thus, we suggest that HsbA is the first protein to be described in a new class. This class of proteins includes the M. anisopliae 4MeS protein (NCBI accession number EAA58613), which has 31% identity to HsbA and is expressed when this entomopathogenic fungus grows on insect cuticle, a naturally hydrophobic material.

The deduced amino acid sequence of HsbA contains a KexB cleavage site before the mature sequence, suggesting that HsbA is synthesized as a preprotein that is secreted through the trans-Golgi network. HsbA was detected only in the extracellular fraction (Fig. 2A) and was not attached to the cell wall after secretion into the culture medium. The RolA hydrophobin of A. oryzae is found on the cell surface of mycelia grown in a liquid medium containing PBSA as the sole carbon source (26), so the localization of HsbA differed from that of RolA.

HsbA bound slightly to the surface of PBSA microparticles in aqueous solution, but the adsorption increased when NaCl or CaCl2 was added (Fig. 3A). Since NaCl is an antichaotropic ion, antichaotropic ions apparently can change the conformation of HsbA and enhance hydrophobic interactions between HsbA and the hydrophobic surfaces of PBSA pellets. When HsbA was adsorbed to the surface of PBSA pellets in the presence of CaCl2, the addition of EDTA released HsbA from PBSA (Fig. 3B). Thus, the calcium ion but not the chloride ion appeared to be critical for adsorption. We did not determine the mechanism of facilitation of the adsorption by calcium ions. Nonionic detergents such as Tween 80 and Triton X-100 also could release HsbA from PBSA pellets, suggesting that HsbA was adsorbed on PBSA surfaces by hydrophobic interactions (Fig. 3B). Since the RolA hydrophobin does not require any ions to attach to PBSA (26), the binding mechanism of HsbA to PBSA appears to differ from that of RolA.

CutL1 more effectively degraded PBSA microparticles precoated with HsbA than those without HsbA (Fig. 5). HsbA does not have esterase activity (data not shown), so HsbA promotes PBSA degradation through an interaction between HsbA adsorbed on PBSA surfaces and CutL1. When the RolA hydrophobin attaches to the PBSA surface, it recruits CutL1, and the accumulation of CutL1 onto the PBSA surface stimulates PBSA hydrolysis (26). When HsbA was attached to a QCM electrode as a ligand, the HsbA molecules had a high affinity to soluble CutL1 (Fig. 5); however, soluble HsbA did not interact with CutL1 adsorbed to a QCM electrode (Fig. 5). The KD value (1.4 x 10–8 M) for interaction between HsbA coated on the QCM electrode and soluble CutL1 (Fig. 5) was similar to that observed for antigen-antibody interactions in general. When anti-HsbA antibody or anti-CutL1 antibody was added to a mixture of CutL1 and soluble-state HsbA in the absence of any hydrophobic materials, essentially no coimmunoprecipitation of HsbA and CutL1 occurred (data not shown), which supports the hypothesis that the form of HsbA adsorbed to PBSA has a higher affinity for CutL1 than does the free form. Promotion of CutL1-dependent PBSA hydrolysis by HsbA based on the biochemical aspects of HsbA would be applicable to solid-state fermentation of PBSA by using A. oryzae cells coexpressing HsbA and CutL1.

We found expressed sequence tags of hsbA in the cDNA library prepared from A. oryzae cells grown in a wheat bran solid-state culture (http://www.nrib.go.jp/ken/EST/db/blast.html). Thus, transcription of hsbA seems to be upregulated in cells grown in the solid-state culture with wheat bran medium (16). The solid-state culture conditions which are preferable to industrial production of many hydrolytic enzymes by A. oryzae are thought to originate from the infection of ancestors of A. oryzae to plant seeds such as rice and wheat (15). Wheat bran medium contains a relatively high concentration of calcium (73 mg calcium per 100 g wheat bran; http://www.ars.usda.gov/main/site_main.htm?modecode=12354500), suggesting that calcium levels in wheat bran medium commonly used for fermentation of A. oryzae are usually >1 mM. These levels seem high enough to promote HsbA adsorption on hydrophobic surfaces (Fig. 3A). A. oryzae grows well on plant cereals, such as wheat bran, over a relatively wide range of water activities (aw = 0.86 to 1) (20); this range includes the aw value corresponding to 0.5 M NaCl (aw = 0.98) that promoted HsbA adsorption to PBSA (Fig. 3A). Therefore, a solid-state culture of A. oryzae cells highly coexpressing HsbA and CutL1 with PBSA as a solid substrate would produce a PBSA mash coated with HsbA and CutL1; subsequent addition of water or appropriate buffers to the mash is expected to facilitate hydrolysis of PBSA. This experiment is now in progress.

In conclusion, we discovered a novel hydrophobic surface binding protein, HsbA, from A. oryzae cells grown in a medium containing PBSA as the sole carbon source and confirmed promotion of PBSA degradation through the interaction with CutL1 on the PBSA surfaces. HsbA is not classified as a hydrophobin, and A. oryzae seems to use several types of surface-active proteins to degrade hydrophobic materials such as biodegradable plastics or natural hydrophobic biomass. We expect our discovery to contribute to the development of large-scale recycling systems for biodegradable plastics or industrial applications for composting hydrophobic biomass.


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ACKNOWLEDGMENTS
 
We thank Toshitaka Minetoki, Motoaki Sano, and Ryoji Ishioka for providing plasmids and PBSA pellets and microparticles; we are grateful to Takafumi Uchida, Youko Yoko, Osamu Mizutani, Takahiro Tanaka, Yoshihiko Matsuda, and Shunsuke Takeuchi for helpful discussions and technical assistance.

This work was supported in part by Innovation Plaza Miyagi of JST (Japan Science and Technology Agency).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumi-dori, Aobaku, Sendai 981-8555, Japan. Phone: 81-22-717-8777. Fax: 81-22-717-8778. E-mail: kabe{at}biochem.tohoku.ac.jp. Back


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Applied and Environmental Microbiology, April 2006, p. 2407-2413, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2407-2413.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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