Note Production of Polygalacturonase by Recombinant Aspergillus Oryzae

Food Sci. Technol. Res., 16 (5), 517–521, 2010 Note

Production of Polygalacturonase by Recombinant Aspergillus oryzae in Solid-State

Fermentation Using Potato Pulp

1 1 1 1 1 Satoshi Suzuki , Mari Fukuoka , Sawaki Tada , Mayumi Matsushita-Morita , Ryota Hattori , 2 1* Noriyuki Kitamoto and Ken-Ichi Kusumoto

1 National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2 Food Research Center, Aichi Industrial Technology Institute, 2-1-1 Shinpukuji-cho, Nishi-ku, Nagoya 451-0083, Japan

Received March 16, 2010; Accepted June 24, 2010

The aim of this study was to produce valuable products from potato pulp, which is a byproduct of potato starch production and contains a high concentration of starch. We used the pulp as a substrate for polygalacturonase (PG) production in solid-state fermentation by a recombinant Aspergillus oryzae strain, PGB3. To generate PGB3, we constructed the PG gene (pgaB) overexpression vector pGBmR, and used that vector to transform the A. oryzae strain, RIB40. PGB3 carries a PG gene under control of the Taka-amylase gene promoter. When cultured in potato pulp solid medium, PGB3 grew more rapidly and produced 4.5 times more PG than RIB40. We observed maximum production (173 U/g) of PG after 2 days of culture of PGB3 in potato pulp solid medium. This high-level PG production from potato pulp demon- strates its potential as a useful substrate in fermentation.

Keywords: potato pulp, polygalacturonase, Aspergillus oryzae, solid-state fermentation, recombinant enzyme

Introduction tive host for heterologous or homologous gene expression. A Conversion of agricultural byproducts into valuable ma- high level of expression of a particular gene requires a strong terials is important for both sustainable agriculture and envi- promoter. The promoter of the amyB gene, which encodes ronmental conservation. Potato pulp is a byproduct of potato the alpha-amylase (Taka-amylase) of A. oryzae, is one of the starch production, with 1 × 105 tons being produced in Japan most inducible promoters of Aspergillus spp. (Tada et al., annually (Oda et al., 2002). The pulp rots rapidly due to its 1991). Because Taka-amylase production is strongly induced high content of nutrients and water, limiting its uses. Al- in starch-containing medium, the amyB gene promoter is though some potato pulp is used as compost or cattle feed in likely to be expressed at high levels in potato pulp. Thus, fer- Japan, much of it is disposed of as industrial waste. Recently mentation of potato pulp by a strain of A. oryzae, with gene there have been several attempts to add value to potato pulp expression controlled by the amyB gene promoter, should be by using it as a substrate for fungal fermentations (Klingspohn an ideal system for producing high-value products such as and Schügerl, 1993; Oda et al., 2002). industrial enzymes. Potato pulp is likely to be a suitable substrate for solid- Microbial pectinases are widely used in industries such state fermentation by the fungus Aspergillus oryzae because as those producing fruit juice and wine. Most of the commer- of its high content of starch (37% of dry matter) (Mayer and cial pectinases produced by Aspergillus spp. contain several Hillebrandt, 1997). A. oryzae is used to produce Japanese kinds of pectinase activity; some examples are polygalactu- fermented foods such as sake, soybean paste (miso) and ronase (PG), pectin methyl esterase and pectin lyase (Lang soy sauce (shoyu). Since A. oryzae produces many different and Dornenburg, 2000). Of these, PG is the most important proteins and secretes many different enzymes, it is an attrac- because it depolymerizes the principal chains of pectin, and is the preferred pectinase for certain foods, such as baby *To whom correspondence should be addressed. foods (Lang and Dornenburg, 2000). Therefore, recombinant E-mail: [email protected] fungal strains that produce a large amount of PG without oth- 518 S. Suzuki et al. er pectinolytic activities are desirable (Lang and Dornenburg, sulting 3’ entry clone without tags was named pg3’S. The 2000). Two PG genes, pgaA and pgaB, have been cloned destination vector pgDB was constructed by modifying an- from A. oryzae and their gene products were characterized. other destination vector, pgDP (Tada et al., 2009), which was PGB is more stable at high temperatures and more resistant digested by SbfI and KpnI. The resulting SbfI/KpnI fragment to protease-induced degradation than PGA (Kitamoto et al., of the ptrA gene was substituted by the PstI/KpnI fragment 1998). Here, we investigated the potential of potato pulp as of the Bm-resistant gene expression cassette, BmR (Suzuki a substrate for PG production. To achieve this, we generated et al., 2009b). pEpgaB was then fused with the 5’ entry clone a recombinant A. oryzae strain that strongly expressed pgaB pg5’Pa (Mabashi et al., 2006), the 3’-entry clone pg3’S and driven by the amyB gene promoter. We investigated the effi- the destination vector pgDB using the MultiSite Gateway LR ciency of PG production by this recombinant A. oryzae strain recombination reaction (Life Technologies Japan Ltd.) ac- using potato pulp as a fermentation substrate. cording to the manufacturer’s protocol. The resulting vector was named pGBmR. Materials and Methods Fungal transformation Transformation of A. oryzae Strains and media A. oryzae strain RIB40 was obtained RIB40 using Ca2+ and polyethylene glycol was carried out from the National Research Institute of Brewing, Japan. For as described previously (Suzuki et al., 2009b). Mycelia of fungal transformation, we used a low-concentration malt A. oryzae RIB40 cultured overnight in YPD medium were extract polypeptone (LMP) medium, composed of 1% malt used to generate protoplasts, which were then incubated with extract and 0.05% polypeptone, with 0.007% Triton X-100, 20 μg pGBmR for 30 min on ice. The transformed proto- 0.1 mM chlorpromazine (Sigma-Aldrich, Tokyo, Japan) and plasts were suspended in molten top agar (0.4% low-melting 30 μg/mL of bleomycin (Bm) sulfate (Cosmo Bio, Tokyo, agarose, 0.8 M NaCl) with potato dextrose broth, and poured Japan). YPD medium (1% yeast extract, 2% polypeptone, onto potato–dextrose agar medium with 0.8 M NaCl. The and 2% glucose) was used for pre-incubation to obtain fun- protoplasts transformed with DNA were incubated for 4 days gal protoplasts. YPS medium (1% yeast extract, 2% poly- on nonselective regeneration agar medium, to allow com- peptone, and 1% starch) was used to induce the amyB gene plete regeneration and sporulation in the absence of Bm. The promoter. Escherichia coli DH5α and Luria-Bertani media resulting conidia were spread at approximately 1.2 × 107 co- were used for the steps involving construction of the plasmid nidia/plate on LMP–agar medium supplemented with 30 μg vector for fungal transformation. Potato pulp solid medium Bm/mL, 0.007% Triton X-100 and 0.1 mM chlorpromazine. (Suzuki et al., 2009a) was prepared by adding water to dried Fermentation We inoculated 500-g lots of autoclaved potato pulp (Kamikawa-Hokubu-Noukyou, Hokkaido, Ja- potato pulp solid medium (60% water content) or wheat bran pan) to give a water content of 60%. Ammonium phosphate solid medium (60% water content) in stainless steel trays (monobasic; Wako, Osaka, Japan) and urea (Wako) were with conidia of A. oryzae RIB40 or the transformants. The added to give a final concentration of 1% each. We used trays were then sealed with aluminum foil and incubated at wheat bran solid medium composed of 40% wheat bran and 30℃. The cultures were mixed once a day. 60% water. Assay of PG activity To assay PG activity, 1 g of either Construction of PG expression vector The vector for potato pulp culture or wheat bran culture was suspended in overexpression of PG in A. oryzae RIB40, was constructed 10 mL of 10 mM sodium acetate buffer (pH 5.0) and incubat- using the MultiSite Gateway Technology (Hartley et al., ed at 4℃ with periodic shaking. PG activity was determined 2000). The necessary entry clones were previously con- by measuring the reducing groups liberated from 0.5% poly- structed using this technology. The coding region of the PG galacturonic acid after incubation of the culture supernatants encoding gene, pgaB (Kitamoto et al., 1998), was ampli- at 37℃ for 15 min in 50 mM sodium acetate buffer (pH 5.0) fied by polymerase chain reaction (PCR) from the genomic using the 3,5-dinitrosalicylic acid (DNS) method (Chaplin, DNA of A. oryzae RIB40 using the primer set (pgaB5dire- 1986), with glucose as a reference. One enzyme unit was topo [5’-CACCATGCATTTCCAACTTCTCGGCCT-3’] defined as the amount of enzyme that produced 1 μmol of and pgaB3 [5’-TTAGCAAGAAGCGCCAGAAGGAAT- reducing groups per minute. GTTC-3’]). The PCR product was cloned into the pENTR/ Determination of fungal cell mass To determine fungal D-TOPO vector (Life Technologies Japan Ltd., Tokyo, Ja- cell mass, a 2-g portion of either potato pulp culture or wheat pan) according to the manufacturer’s protocol. The resulting bran culture was suspended in 10 mL of 50 mM phosphate entry vector was named pEpgaB. The 3’ entry clone, pg3’HH buffer (pH 7.0) containing 5% takadiastase (Sankyo, Tokyo, (Mabashi et al., 2006), was digested by SmaI and religated Japan), 0.1% cellulosin PE60 (HBI Enzymes Inc., Shiso, by self-ligation to exclude epitope tag sequences. The re- Japan) and 0.1% cellulosin HC100 (HBI Enzymes Inc.). The Production of Pectinase in Potato Pulp 519 suspension was incubated at 42℃ overnight with shaking, to strains that are able to grow on potato pulp solid medium. degrade the plant tissue and thus make sample handling eas- Comparing the sequence of the pgaB gene of strain ier. The cell mass of A. oryzae in the culture was determined RIB40 with the original pgaB gene of strain KBN616, we according to the procedure of Fujii et al. (1992). The culture found that two base substitutions (G to C at 472 and A to sample treated by the commercial enzymes was washed three C at 520) in the nucleotide sequence led to two amino acid times with 10 mL of 50 mM phosphate buffer (pH 7.0) and substitutions (G to A at 136 and Y to S at 152) in the deduced then re-suspended in 10 mL of the same buffer. To liberate amino acid sequence. PGB was expected to be a monomer N-acetylglucosamine (GlcNAc) from the fungal cell wall, enzyme, because the orthologue in A. niger, PGI, is a mono- 10 mg of yatalase (Takara Bio, Otsu, Japan) was added to mer enzyme (Bussink et al., 1991). Four disulfide bonds the sample suspension, and the sample suspension was incu- were predicted in PGB by the online software, DISULFIND bated at 37℃ for 1 h. The amount of GlcNAc in 500 μL of (Ceroni et al., 2006). Transformants grown in YPS medium supernatant of the sample was determined according to the for a day were assayed for starch-inducible PG activity to se- procedure of Reissig et al. (1955). lect a strain with the capacity to produce a high level of PGB for further study. Among the 10 transformants screened, we Results and Discussion identified one transformant, named PGB3, that produced the Generation of recombinant A. oryzae strain Recombi- highest level of PGB. nant A. oryzae with strong expression of the PG gene was Growth curves of the strain PGB3 We examined the constructed as described. Because potato pulp still contains ability of strains RIB40 and PGB3 to grow in potato pulp starch, we employed a starch-inducible high-expression solid medium, and compared this with their ability to grow promoter, the amyB gene promoter, which is a part of the in wheat bran solid medium, a medium commonly used for 5’-upstream regulatory sequences of the gene encoding commercial fungal enzyme production (Pandey, et al., 1999). Taka-amylase, to control expression of the pgaB gene and Figure 3 shows fungal cell mass for up to 3 days. Strain thus production of PGB, one of the two PGs of A. oryzae PGB3 exhibited a significantly higher growth rate in potato (Fig. 1). Although the original pgaB was cloned from the pulp solid medium than in wheat bran solid medium. This A. oryzae strain, KBN616, for soy sauce production (Kita- growth rate is similar to that observed for the host strain moto et al., 1998), we cloned the gene from strain RIB40 RIB40 in both medium types. (AO090023000401). This strain is known to grow on potato Potato pulp dry matter contains 37% starch, 17% cel- pulp solid medium (Suzuki et al., 2009a). Moreover, genetic lulose, 14% hemicellulose and 17% pectin (Mayer and manipulation is easier in this strain than in the other A. oryzae Hillebrandt, 1997). Starch is easily degraded to glucose by α-amylase and glucoamylase, and A. oryzae is able to se- crete large amounts of amylases into its culture medium. However, most of the starch in potato pulp is located within intact starch cells that are protected by a cell wall covered with sticky pectic substances (Mayer and Hillebrandt, 1997). Saito et al. (2003) revealed a positive correlation between PG activity and lactic acid productivity in lactic acid fermentation by Rhizopus oryzae using potato pulp as the substrate (i.e., the more R. oryzae produced PG, the more efficiently the fungus also produced lactic acid). Moreover, the addition of a commercial pectinase to potato pulp culture inoculated with fungal spores improves the efficiency of lactic acid fermentation of R. oryzae strains (Saito et al., 2003). PG produced by strain PGB3 could degrade the pectic substances covering the cell wall of the starch cells in potato pulp and thus allow amylases to access this starch source. If this is the case, PGB3 would use more glucose than the Fig. 1. Plasmid map of the pgaB gene overexpression vector controlled by the amyB gene promoter. host strain RIB40 in the potato pulp solid medium. In our Ampr, β-lactamase gene; bp, base pairs; BmR, bleomycin resistant previous study, we showed that A. oryzae strain NFRI1163 gene cassette; PamyB, amyB gene promoter; TamyB, amyB gene consumed most of the starch in potato pulp solid medium in terminator. 5 days of culture (Suzuki et al., 2009a). Because of its higher 520 S. Suzuki et al.

200 6 RIB40 (W.B.) RIB40 (W.B.) 180 PGB3 (W.B.) PGB3 (W.B.) 5 160 RIB40 (P.P.) RIB40 (P.P.) 140 PGB3 (P.P.) PGB3 (P.P.) 4 120

100 3 80

2 60 Pgase activity (U/ g media) Cell mass (mg/ g media) 40 1 20

0 0 0 1 2 3 0 1 2 3 Cultivation time (days) Cultivation time (days)

Fig. 2. Growth rate of strain PGB3 and RIB40. Fig. 3. Polygalacturonase production of solid-state fermentation The graph shows averages of the values from duplicate experi- by the strain PGB3 and RIB40. ments. P.P., potato pulp; W.B., wheat bran. Cell mass was indicated The experiment was duplicated and the average value from two ex- by milligrams of cell mass per gram of solid medium. periments is presented. Pgase, Polygalacturonase; P.P., potato pulp; W.B, wheat bran. Polygalacturonase activity is indicated by units per gram of solid medium. PG activity, PGB3 is expected to degrade starch into glucose more quickly than NFRI1163. We observed that the cell mass of PGB3 in potato pulp solid medium was still increasing better in potato pulp culture than in the wheat bran culture after 9 days of culture (data not shown). Therefore, it seems (Fig. 3). This suppression of enzyme activity might have that almost all the starch was converted to glucose within a been caused by glucose repression of the amylase promoter. few days and that the fungi used glucose as a carbon source As mentioned above, it is possible that glucose is abundant until at least day 9 of culture. in potato pulp solid medium after a few days culture because PG production by potato pulp fermentation We evalu- of starch degradation by amylases secreted by A. oryzae. A ated the level of production of PG by the fermentation of further increase in the production efficiency of PG in this potato pulp solid medium by PGB3 for up to 3 d of culture system might be possible if repression of the amyB gene pro- and compared this level with that produced by the fermenta- moter by glucose in A. oryzae can be overcome. tion of wheat bran solid medium (Fig. 3). Maximum production of PG was obtained after 2 d of culture of PGB3 in Conclusions both potato pulp solid medium (173 U/g solid medium) and We were able to obtain efficient production of PG using wheat bran solid medium (140 U/g solid medium). Further potato pulp as a substrate for fungal fermentation. Potato culture resulted in a gradual decrease in enzyme activity for pulp was the preferred substrate for PG production in solid- up to 10 d (data not shown). By comparison, the PG produc- state fermentation by genetically modified A. oryzae, in tion profiles of the host strain RIB40 showed that maximum which the amyB gene promoter was effectively functional. production was attained after 1 d of culture and either stayed at that level (in potato pulp solid medium) or decreased to Acknowledgements We thank Prof. Kitamoto and Dr. J.-I. the baseline level (in wheat bran solid medium) by day 3. Maruyama (University of Tokyo) for providing the plasmid pg5’Pa The enzyme activity of PGB3 was notably higher than that and pg3’HH. This work was supported by a grant from the Ministry of RIB40 in both medium types for up to 3 d of culture. The of Agriculture, Forestry and Fisheries of Japan (Rural Biomass Re- enzyme activities of both strains were comparable or bet- search Project, Biomass Material BM-D2110). ter in potato pulp solid medium than in wheat bran solid medium. PGB3 grew in potato pulp solid medium approxi- References mately twice as fast as in wheat bran solid medium (Fig. Bussink, H.J.D., Brouwer, K.B., de Graaff, L.H., Kester, H.C.M. 2), although the enzyme activity of PGB3 was only slightly and Visser, J. (1991). Identification and characterization of a sec- Production of Pectinase in Potato Pulp 521

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