Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae
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- Sato, H., Toyoshima, Y., Shintani, T. et al. Appl Microbiol Biotechnol (2011) 92: 961. doi:10.1007/s00253-011-3422-0
We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.
KeywordsAspergillus oryzaeα-AmylaseCell wallChitinSubmerged cultureProtein production
Aspergillus oryzae has been used in fermentation industries in Japan for, e.g., production of sake and soy sauce, for over a thousand years to produce copious amounts of amylolytic enzymes. Among the amylolytic enzymes produced by A. oryzae, α-amylase (Taka-amylase A; TAA) is produced in the largest amounts. When the production of TAA by A. oryzae was analyzed in a submerged culture medium, we observed that TAA activity disappeared in the later stage of cultivation. In heterologous protein production, proteins are sometimes degraded by extracellular proteolytic enzymes that are secreted into the medium. In fact, protease gene-disrupted mutants show a notably increased productivity of heterologous proteins (Jin et al. 2007; Yoon et al. 2009, 2011). However, the disappearance of TAA did not result from protein degradation but was caused by TAA adsorption onto the surface of the mycelium.
It was documented nearly five decades ago that TAA accumulates onto the surface of fungal cell wall as well as in the medium (Tonomura et al. 1962, 1963; Tonomura and Tanabe 1964). It was recently reported that TAA was trapped in the cell wall of the mycelium in submerged cultures (Oda et al. 2006) and also that TAA bound to the fungal cell wall could be liberated in an alkaline environment (pH > 7.2) or in a solution containing higher concentration of anions (Tonomura et al. 1963). Although the phenomenon of TAA adsorption onto the cell wall of A. oryzae has been previously reported, the cell wall factor(s) that allows TAA adsorption needs to be identified and the adsorption mechanism behind this remains to be elucidated.
Fungal cell walls mainly comprise polysaccharides such as α-glucan, β-glucan, and chitin (Bartnicki-Garcia 1968; Wessels and Sietsma 1981; Latgé and Calderone 2006). The polysaccharides in fungal cell walls are classified into two groups according to their solubility in alkaline solutions. Alkali-soluble polysaccharides are composed of α-1,3-glucan and galactomannan. In Aspergillus fumigatus, α-1,3-glucan is the major component of the polysaccharides found in this species, accounting for more than 40% of the cell wall (Fontaine et al. 2000). Conversely, the main components of alkali-insoluble polysaccharides are β-1,3-glucan and chitin, both of which constitute the structural skeleton of the cell wall. Chitin is a long, linear polymer of β-1,4-linked N-acetylglucosamine. It is the major structural polysaccharide found in cell walls of filamentous fungi, accounting for 10–20% of the cell wall, and contributes significantly to the integrity of the cell wall (Bowman and Free 2006). β-1,3-glucan is another major structural component of the cell wall and is highly branched with β-1,6 linkages. Chitin, galactomannan, and β-1,3/1,4-glucan are covalently bound to β-1,3-glucan. The alkali insolubility of β-1,3-glucan is due to its covalent linkages with chitin (Hartland et al. 1994).
The A. oryzae cell wall allows not only TAA adsorption but also heterologous protein, such as bovine serum albumin (BSA) and hen egg lysozyme, adsorption (unpublished results; Yabuki and Fukui 1970). A. oryzae is an attractive host for the production of commercially useful heterologous proteins due to its ability to secrete large amounts of enzymes (Christensen et al. 1988; Tsuchiya et al. 1992, 1993). However, it is difficult to obtain a high yield as expected because there is only limited information available on the protein secretion mechanism. Therefore, protein adsorption onto the cell wall could have deleterious effects on the productivity of heterologous proteins in A. oryzae. It is necessary to understand the phenomenon of protein adsorption to achieve high levels of protein production in A. oryzae. In this study, the cell wall was fractionated by alkali extraction and enzymatic digestion to identify the component(s) that allows TAA adsorption onto the cell wall of A. oryzae.
Materials and methods
Strains and media
A. oryzae strains used in this study were RIB40 (wild-type strain; National Research Institute of Brewing, Higashihiroshima, Japan) and AOK11 (industrial strain; Akita Konno Shoten, Akita, Japan). Conidiospores were obtained from malt agar medium (2% malt extract, 1% polypeptone, 2% glucose, and 1.5% agar) after 4–6 days of cultivation at 30°C. For fungal growth and TAA production, YPM medium containing 0.5% yeast extract, 1% Bacto Peptone, and 2% maltose was used. Approximately 106 conidiospores per 50 ml of medium were inoculated and incubated at 30°C with shaking at 120 rpm.
The activity of TAA was measured using the iodine–starch method, as previously described (Adachi 1954). In brief, 100 μl of the culture broth or supernatant of the mixture from TAA binding assay was added to 2 ml of 1% soluble starch in a 40-mM sodium acetate buffer (pH 5.0) prewarmed for 5 min at 40°C, and the mixture was incubated at 40°C. After incubation for an appropriate period, 20 μl of the reaction mixture was retrieved and added to 2 ml of iodine solution (1.25 mM I2, 6 mM KI in 0.5% HCl), and the transmissivity of the resulting solution was spectrophotometrically measured at 670 nm. One unit of TAA activity was defined as the amount of enzyme required to digest starch until the transmissivity of 66% of the iodine–starch solution was given in 30 min under the reaction conditions. The protein concentration was determined by the BCA method using a Pierce BCA Protein Assay Kit—Reducing Agent Compatible (Takara Bio, Otsu, Japan). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of TAA was performed using the method described by Laemmli (1970).
Preparation of cell wall fractions
The mycelium was cultivated in liquid YPM medium, collected by filtration, washed several times with distilled water (DW), pulverized in liquid nitrogen, and suspended in a homogenized solution (HS; 20 mM Tris–HCl, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM tosyl lysine chloromethyl ketone, 1 mM tosyl phenylalanine chloromethyl ketone, 1 mM leupeptin, 1 mM pepstatin, 1% Triton X-100; pH 7.6). The pellets obtained by centrifugation at 3,000×g were washed eight times with HS, and the precipitate was used as the initial disrupted cell wall (CW1). The CW1 fraction was incubated in 0.1 M NaOH at room temperature for 30 min, followed by centrifugation at 3,000×g. The precipitate was then lyophilized after washing three times with HS to yield the dilute alkali-treated cell wall fraction (CW2). Subsequently, the CW2 fraction was incubated in 1 M NaOH at room temperature for 30 min, followed by centrifugation at 3,000×g, and the resulting precipitate was lyophilized after washing three times with HS to yield the alkali-treated cell wall fraction (CW3). Finally, the CW3 fraction was boiled in 1 M NaOH for 15 min, centrifuged at 3,000×g, and the resulting precipitate was lyophilized after washing three times with HS to yield the hot alkali-treated cell wall fraction (CW4).
Determination of TAA adsorption capacity of cell wall fractions
Four milligram of each lyophilized cell wall fraction was suspended in 2 ml of 10 mM phosphate buffer (pH 5.0) and 0.5 ml TAA (α-amylase from A. oryzae; Sigma-Aldrich, St. Louis, USA) dissolved in 10 mM phosphate buffer (pH 5.0; at a concentration of 4 mg/ml) was added to the suspension. The mixture was gently shaken at room temperature for the indicated incubation period and then centrifuged to collect the supernatant for TAA analysis. The TAA adsorption capacity, represented as the amount of TAA adsorbed onto the cell wall, was calculated by subtracting the amount of TAA remaining in the supernatant from the total amount added.
To examine the TAA adsorption capacity of the polysaccharides, 4 mg of powdered chitin from crab shells (Wako Pure Chemicals Industries, Osaka, Japan) or curdlan from Alcaligenes faecalis (Wako Pure Chemicals Industries, Osaka, Japan) was added to 2.5 ml of 10 mM phosphate buffer (pH 5.0) containing 0.8 mg/ml TAA.
Enzymatic treatment of cell wall fractions
One hundred and fifty milligrams of CW4, the hot alkali-treated cell wall fraction, was digested in 15 ml of 66 mM phosphate buffer (pH 7.0) containing 2 mg Zymolyase-100T from Arthrobacter luteus (Seikagaku Corp., Tokyo, Japan) or 100 mM phosphate buffer (pH 6.0) containing 5 mg chitinase from Bacillus sp. (Wako Pure Chemicals Industries, Osaka, Japan) at 37°C for 2 days. Proteolytic treatment was performed using 2 mg proteinase K (Sigma-Aldrich, St. Louis, USA) for 1 h under conditions similar to Zymolyase-100T digestion. After incubation, the precipitate was washed three times with DW and then repeatedly digested with the same enzyme. The resulting pellets were washed and lyophilized.
Determination of chitin content
Five milligrams of the lyophilized cell wall fraction was suspended in 0.4 ml of 4 N HCl and hydrolyzed at 96°C for 12 h, followed by centrifugation to remove any insoluble material. The liberated glucosamine in the resulting supernatant was determined by the Elson–Morgan reaction as modified by Blix (1948). Briefly, an aliquot of the supernatant was added to 0.4 ml acetylacetone reagent (1.25 M Na2CO3 in 3% [v/v] acetylacetone) and incubated at 90°C for 1 h. Then, 4 ml ethanol and 0.4 ml Ehrlich’s reagent (1.6 g p-dimethylaminobenzaldehyde in 30 ml of HCl and 30 ml of ethanol) was added and incubated for 1 h at room temperature. The absorbance at 530 nm was measured using glucosamine as the standard.
Adsorption of TAA onto the surface of the fungal cells grown in a submerged culture
We previously determined that TAA was produced by host strains, including A. oryzae NS4, in heterologous protein production (Yamada et al. 1997), but this ability disappears in the later stage of cultivation in submerged cultures. Therefore, to examine whether the disappearance of TAA during cultivation is observed in other A. oryzae strains, wild-type RIB40 and industrial AOK11 strains were grown in a liquid medium (YPM) for TAA production, which was monitored by measuring TAA activity and protein levels in the culture broth. High TAA activity and protein production were observed in the culture broth of both strains at 24 h of cultivation, but thereafter TAA activity decreased rapidly concomitant with the disappearance of TAA (Supplemental Fig. 1). This result indicates that the disappearance of TAA generally occurs in the later stage of submerged cultivation of A. oryzae.
Adsorption of TAA onto disrupted cell wall preparations
Alkali fractionation of cell wall preparations
The cell walls of filamentous fungi, including A. oryzae, consist of polysaccharides (over 90% of the cell wall) and small amounts of proteins. To examine whether proteins bound to the cell wall are involved in TAA adsorption, cell walls with high TAA adsorption capacity were boiled before binding assay was performed. The adsorption capacity was not affected by boiling treatment for 20 min (data not shown), suggesting that cell wall component(s) that efficiently allows TAA adsorption is a polysaccharide and not a protein.
Enzymatic treatment of alkali-insoluble cell wall fraction
Chitin content of the hot alkali-treated cell wall fraction digested with enzymes
Chitin content (μg/mg dry cell wall)
224 ± 1.8
498 ± 8.5
69 ± 1.1
TAA adsorption capacity of the cell wall in the early stage of cultivation
Chitin content of alkali-treated cell wall fractions harvested at the early and later stages of cultivation
Cell wall fraction
Chitin content (μg/mg dry cell wall)
166 ± 6.0
311 ± 10
361 ± 51
310 ± 63
133 ± 3.0
283 ± 18
328 ± 28
323 ± 27
Chitin content of the cell wall fraction CW124-h following proteolytic and chemical treatments
Chitin content (μg/mg dry cell wall)
142 ± 10
125 ± 29
172 ± 30
209 ± 21
0.1 M NaOH (CW224-h)
274 ± 48
We observed that TAA secreted once in the early stage of submerged culture is adsorbed onto the mycelium in the later stage of cultivation. The observation that TAA is bound to the surface of the fungal cell wall had been previously reported (Tonomura et al. 1962, 1963), but the cell wall component(s) responsible for TAA binding remains to be determined. In the present study, treatment of the cell wall fraction with an alkaline solution and enzymes demonstrated that the chitin present in the cell wall is possibly involved in the adsorption of TAA.
We assumed that such a component(s) may be a cell wall protein induced following extended periods of cultivation because the adsorption of TAA onto the cell is not observed in the early stage of cultivation. The immunoprecipitation method with the anti-TAA antibody was used in an attempt to identify the cell protein that interacts with TAA. However, no proteins could be isolated that interacted with TAA in the cell wall (unpublished results). According to a previous report (Tonomura et al. 1963), it is unlikely that cell wall proteins are involved in the adsorption of TAA. Next, polysaccharides that constitute over 90% of the cell became the focus of this investigation, and the fractionated cell walls were treated in a stepwise manner with alkali solutions. Finally, chitinase digestion of the hot alkali-treated cell wall fraction, CW460-h, resulted in a significant decrease in the TAA adsorption capacity of the resulting fraction, indicating that chitin is most likely the main component responsible for the adsorption of TAA. This was further confirmed by the observation that chitin prepared from crab shells also allow TAA adsorption in comparable amounts to the alkali-treated cell wall.
It was reported that not only homologous proteins, such as TAA, but also heterologous proteins, such as BSA and hen egg lysozyme, could be adsorbed onto the cell wall (Yabuki and Fukui 1970). However, TAA does not possess any structural domains that could bind to polysaccharides. Moreover, TAA, BSA, and lysozymes share no structural similarities. The adsorption mechanisms of these proteins are not clear. Chitin is a linear polysaccharide consisting of repeating N-acetylglucosamine units, some of which are deacetylated. The deacetylated glucosamine content generally varies between 8% and 15% in chitin. The primary amine group of glucosamine makes the chitin polymer a positively charged polysaccharide under acidic conditions; thus, the resulting chitin could only interact with negatively charged proteins. Since TAA binds to the mycelium or the cell wall at acidic pH and is liberated at alkaline pH, as well as at higher buffer concentrations, which was demonstrated in the present study (Fig. 2) and in a previous study (Tonomura et al. 1963), it may be possible that the adsorption of TAA onto chitin is caused by ionic interactions. In this regard, it would be interesting that the effect of N-deacetylation and N-acetylation treatments of crab chitin on the TAA adsorption capacity is further investigated. The mechanism for the TAA adsorption onto chitin would be different from the mechanism for the cell surface protein that binds to chitin in the dimorphic fungus Blastomyces dermatitidis (Brandhorst and Klein 2000). In B. dermatitidis, the protein adhesin WI-1 is localized to the cell surface through specific binding to chitin. However, half of WI-1 on the cell surface can be released by water washes (Brandhorst and Klein 2000), which is in clear contrast to the observation in this study that TAA bound to the cell wall could not be liberated with water or at low concentrations of buffer.
Although the cell wall component that adsorbs TAA is most likely chitin, which is one of major components of the cell wall in A. oryzae and assumed to be of sufficient quantity in the cell wall regardless of the culture period, the ability to adsorb TAA only appeared in the later stage of cultivation, but not in the early stage. There is a report that describes the TAA adsorption capacity of A. oryzae when grown in a liquid medium in which little TAA was produced, but not when grown in a starch-containing medium (Yabuki and Fukui 1970). In contrast, we observed the adsorption of TAA onto the mycelium in liquid YPM medium. Yabuki and Fukui (1970) proposed the existence of a masking factor (protein) that masked the TAA binding site of the cell wall and concomitantly promoted TAA induction. The observations of this study may be explained as follows: the masking factor(s) is produced in the early stage of cultivation in the YPM medium, resulting in the prevention of TAA adsorption onto the mycelium. In contrast, in the later stage of cultivation, the masking factor is not produced due to the depletion of maltose, which is an inducer of TAA production, resulting in the decrease of masking factor levels and in the appearance of the TAA adsorption capacity of the mycelium. However, when treated with dilute alkali, the initial disrupted cell wall, CW124-h, which initially had no TAA adsorption capacity, exhibited a high TAA adsorption capacity. In addition, the digestion of CW124-h with proteinase K did not significantly affect the TAA adsorption capacity. These observations indicate that the putative masking factor that prevents the TAA adsorption onto the mycelium is not a protein, as reported by Yabuki and Fukui (1970). In addition, although the mycelium in the later stage of cultivation (e.g., after 60 h culture) had a high TAA adsorption capacity, the stepwise alkali fractionation of the cell wall showed that the cell wall fraction obtained by dilute alkali treatment exhibited a much higher TAA adsorption capacity than the disrupted cell wall fraction, as shown in Fig. 4. This suggests that the putative inhibiting factor still exists, to some extent, in the cell wall at later stages of cultivation. If there is such an inhibiting factor(s) for the TAA adsorption to the cell wall, it may be an ionic compound because the TAA adsorption to chitin is probably caused by ionic interactions. Alternatively, since the TAA adsorption capacity was exhibited by the dilute alkali treatment of CW124-h, a potential candidate for the inhibiting factor could be α-1,3-glucan since it is a polysaccharide in the cell wall that can be extracted with dilute alkali. Future research is aimed at identifying such an inhibiting factor.