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.