Strains and media
Strains H. fabianii J640 and Cryptococcus sp. S-2 were obtained from the National Research Institute of Brewing culture collection, Japan. A uracil auxotrophic mutant of H. fabianii J640, named H. fabianii J640 u-1, lacking orotidine-5'-phosphate decarboxylase, was used as a host strain for new expression vector pHFGE-1. S. cerevisiae YPH-499 (MATα ura3 lus2 ade2 trp1 his3 leu2) was used as the host for transformation vector pG-1 (Schena et al. 1991). E. coli strain HB101 and JM109 were employed as the host of plasmid vector, which were used for DNA manipulation and construction of the gene library.
Yeast cells were grown on YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose) and YPD medium (1% yeast extract, 2% peptone, 2% glucose). Luria-Bertani medium containing ampicillin (100 μg/ml) was used to cultivate E. coli. The minimal medium containing 1% glucose and 0.67% yeast nitrogen base (YNB) without amino acids was used to select the yeast transformants. YPM medium was prepared by replacing the glucose of YPD with maltose. The medium used to investigate expression induction, contained 1% yeast extract, 1% casamino acid, and 2% glucose or maltose.
Expression vector for H. fabianii J640
The expression vector pHFGE-1 (Kato et al. in press) (Figure 1A) was used. The cloning site of this vector is a Bam HI site between the promoter and terminator from H. fabianii J640 glucoamylase DNA. The host cell of this vector is a uracil auxotrophic mutant designated as H. fabianii J640 u-1, and it could be transformed by a non-homologous and frequently multicopy integration into the host genomic DNA.
Transformation of yeast
Transformations were carried out by electroporation as described by (Becker et al. 1991). Electroporation was done with a Gene Pulser (Bio-Rad) with settings of 200 V and 25 μF using a 0.2 cm cuvette.
Assay of xylanase and α-amylase activity
Xylanase activity was assayed by measuring the amount of reducing sugar liberated from xylan (Iefuji et al. 1996b). One unit of activity was defined as the amount of xylanase needed to liberate 1 μmol of D-xylose per min under the condition just described.
α-Amylase activity was measured with an α-amylase kit (Kikkoman). One unit of α-amylase activity was defined as the amount of enzyme which forms 1 μmol of 2-choloro-4-nitrophenol from 2-choloro-4-nitrophenyl 65-azide-65-deoxy-β- maltopentaoside under the condition described above.
Preparation of model wastewater and treatment test
Model wastewater containing soluble starch was made with 1% refined starch (Merck) and 0.25% yeast extract, pH 6.0. The starch was solubilized by autoclaving. Model wastewater containing insoluble starch was made with 0.25% yeast extract, pH6.0, autoclaved and cooled to approximately 55°C. The same amount of starch was sterilized in 70% ethanol. The suspension was centrifuged and decanted. The starch pellet was then added to the autoclaved yeast extract solution.
Yeast cells were incubated at 30°C for 2 days on YM medium. Then 5 × 106 cells/ml was inoculated to the model wastewater in an Erlenmeyer flask. Cultures were incubated at 30°C with shaking at 105 rpm and samples were periodically harvested.
Yeast cells in the model wastewater were counted with a hemocytometer
The model wastewater containing soluble starch was centrifuged at 3000 rpm for 10 min, and chemical oxygen demand (COD) of the supernatant was measured. The decrease in COD of the model wastewater containing soluble starch was used to express the capacity of the yeast to treat the wastewater.
It was not possible to measure COD of the model wastewater containing insoluble starch because of the difficulty in separating the cells and insoluble starch. In this case, degradation of the starch was measured with the iodo-starch reaction (Sato et al. 1987) as follows: 1 ml culture was heated in a micro tube at 100°C for 30 min to solubilize the starch. Yeast cells were then removed by centrifugation. Iodic liquid (0.2 ml; containing 0.0317 g iodine, 0.1 g potassium iodide and 5 ml 3N-HCl in 100 ml water) was added to the supernatant and the optical density was measured at 670 nm. Transmittance at 670 nm was taken as a measure of starch degradation.
Monitoring the presence of a foreign gene in a transformant
The transformants were cultured in 10 ml YM medium and harvested by centrifugation. DNA was extracted with an Easy-DNA kit (Invitrogen) and used for the PCR template. Unique PCR primers were designed, and the position of the PCR product is shown in Figure 1B. PCR cycling conditions were followed by 25 cycles of 94°C for 1 min, 55°C for 2 min, 72°C for 3 min.
To determine the sensitivity of the PCR, cells were cultured in YM medium, and the cell density was measured. Then a dilution series was made (106-101 cells/ml). One ml of each dilution was harvested and DNA was extracted with the EASY-DNA kit and used as a PCR template.