Construction of expression vectors
The plasmid pET-16b/YGL157w was constructed for production of K. marxianus YGL157w protein with a N-terminal hexahistidine tag. After preparation of genomic DNA from K. marxianus strain DMB1 (strain number: HUT7412), the YGL157w gene (accession number: LC016711) was amplified using PCR with KOD -plus- DNA polymerase (Toyobo, Osaka, Japan) and the primers 5′-CAT
ATGACGTACGTTGTGGTTACTGGTGC-3′ (the NdeI site is in bold and the initiation codon is in italics) and 5′-GGATCC
TTAGTTGTTAGCCTTTAGTATTTG-3′ (the BamHI site is in bold and termination codon is in italics). The PCR product was cloned into pTA2 (Toyobo, Osaka, Japan) and sequenced to check for PCR errors. The YGL157w gene was then excised from the resulting plasmid using NdeI and BamHI and subcloned into pET-16b (Novagen, Hessen, Germany) to give pET-16b/YGL157w.
Expression of proteins
YGL157w protein was expressed in E. coli BL21 (DE3) cells harboring pET-16b/YGL157w and then purified to homogeneity. The cells were grown at 37°C for 3 h in Luria-Bertani (LB) medium (1 L) containing 100 mg/L ampicillin. After inducing expression by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM, the culture was incubated for an additional 3 h. The cells were then harvested, suspended in 20 mM Tris–HCl buffer (pH 7.9) containing 500 mM NaCl (buffer A) and 5 mM imidazole, and disrupted by ultrasonication. The resultant lysate was clarified by centrifugation (27,500 × g for 15 min at 4°C), after which the supernatant was applied to a Chelating Sepharose Fast Flow column (20 mL; GE Healthcare, Buckinghamshire, UK) charged with Ni2+ and equilibrated with buffer A containing 5 mM imidazole. After washing the column with buffer A containing 5 mM imidazole (40 mL) and then 60 mM imidazole (60 mL), the recombinant YGL157w protein was eluted with buffer A containing 500 mM imidazole. The active fractions were pooled, concentrated using a Vivaspin 20 concentrator (10,000 MWCO, Sartorius AG, Goettingen, Germany) and loaded onto a HiLoad 26/60 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) containing 50 mM NaCl. The active fractions were pooled and dialyzed against 20 mM Tris–HCl buffer (pH 7.2). Finally, the dialysate was concentrated and the resultant solution was used for biochemical experiments.
Protein concentrations were determined using the Bradford method with bovine serum albumin (BSA) serving as the standard (Bradford 1976).
Molecular mass determination
SDS-PAGE was carried out on a 10% polyacrylamide gel using the method of Laemmli (1970). EzStandard PrestainBlue (ATTO, Tokyo, Japan) was used as the molecular mass standards. The protein sample was boiled for 5 min in EzApply (ATTO). Protein bands were visualized by staining with EzStainAqua (ATTO).
The molecular mass of the native enzyme was determined by gel filtration column chromatography using a Superdex 200 Increase 10/300 GL column. Conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) served as molecular standards (GE Healthcare).
Assay of enzyme activity
KmGRE2 activity was measured by monitoring the decreases in the absorbance at 340 nm caused by the reduction of aldehyde, or the increases in the absorbance caused by the oxidation of alcohol. The mixture (1 mL) used for the reductive reaction contained 100 mM acetate buffer (pH 5.5), 5 mM aldehyde, 0.2 mM NADPH and YGL157w protein. The mixture (1 mL) used for the oxidative reaction contained 100 mM bicarbonate-NaOH (pH 10.0), 5 mM alcohol, 1.25 mM NADP+ and YGL157w protein. The reaction was started by the addition of coenzymes, and the absorbance at 340 nm was monitored at 25°C using a Shimadzu UV-2450 (Kyoto, Japan). The extinction coefficient of NADPH was 6.22 mM−1 cm−1. One unit of enzyme was defined as the amount of enzyme producing 1 μmol of NADPH per min at 25°C in the reductive reaction of methylglyoxal.
Effects of pH and temperature on enzyme activity
The pH dependence of the reduction catalyzed by YGL157w protein was determined at 25°C using 100 mM concentrations of acetate (pH 4.0–5.5) and citrate (pH 5.5–6.5). The temperature dependence was evaluated by measuring the reductive reaction at temperatures ranging from 25 to 45°C.
Effects of pH and temperature on enzyme stability
The effect of pH on enzyme stability was evaluated by incubating 100 nM YGL157w protein for 30 min at 35°C with 50 mM concentrations of acetate (pH 5.0–5.5), citrate (pH 5.5–6.5), phosphate (pH 6.5–8.0), borate-NaOH (pH 8.0–9.0) and bicarbonate-NaOH (pH 9.0–11.0). The enzyme solution was then rapidly cooled on ice, and the remaining activity was determined using the standard reduction assay. The thermal stability was determined by incubating YGL157w protein in 20 mM Tris–HCl buffer (pH 7.2) for 30 min at temperatures ranging from 25–45°C. The enzyme solution was then rapidly cooled on ice, and the remaining activity was determined using the standard reduction assay.
Determination of kinetic parameters
The initial velocity of the reductive reaction was analyzed using the standard assay conditions. To determine the kinetic constants for methylglyoxal and NADPH, several concentrations of methylglyoxal (0.05–15 mM) or NADPH (0.01–0.15 mM) were used. The initial velocity was then plotted against the substrate concentration, and the K
m and k
cat values were determined by curve fitting using Igor Pro ver. 3.14 (WaveMetrics, Tigard, OR, USA).
Preparation of hydrolysate
Lignocellulosic biomass material (Japanese cedar) was milled using a cutter mill (MKCM-3; Masuko Sangyo, Saitama, Japan), after which the resulting particles were used as the initial raw material. According to Lee et al. (2010), mechanochemical and hydrothermal pretreatment was carried out. The resulting sample was hydrolyzed using 20 FPU/g of Acremonium cellulase (Meiji Seika Pharma, Nagoya, Japan) and 40 μL/g of Optimash BG (Genencor International, Rochester, NY, USA) in 50 mM citrate buffer (pH 5.0) at 50°C and 150 rpm. After incubation for 48 h, the reaction mixture was harvested by centrifugation, and the supernatant was filtered through a 0.2 μm filter (Merck Millipore, Billerica, MA, USA). The pH of the mixture was then adjusted to 6.5, the mixture was diluted, and the resulting solution was used as the hydrolysate. Further details of the procedure are provided elsewhere (Akita et al. 2015).
Effect of KmGRE2 expression on cell growth
The effect of KmGRE2 expression was evaluated by cultivation in a test tube using 3 mL of hydrolysate containing 0.5 mM IPTG, which was incubated at 37°C and 180 rpm. E. coli BL21 (DE3) cells harboring pET-16b/YGL157w or pET-16b were pregrown overnight and then diluted 1:100 with fresh hydrolysate. Cultures were monitored for cell growth at OD600 using an Eppendorf BioSpectrometer (Eppendorf, Hamburg, Germany).
Quantification of sugars and aldehydes
After clarifying the culture by centrifugation and filtration, the supernatant was subjected to high performance liquid chromatography (HPLC). Quantification was performed using an Aminex HPX-87H cationic exchange column connected to an Aminex 85H Micro-Guard Column (Bio-Rad Labs, Richmond, CA, USA). The chromatographic conditions for sugars and aldehydes were as follows: mobile phase, 4.5 mM H2SO4 or 8 mM H2SO4; flow rate, 0.6 mL · min−1; and the column oven temperature, 65°C or 35°C. Sugars and aldehydes were detected using a Jasco RI-2031 Plus Intelligent Refractive Index Detector (Jasco, Tokyo, Japan) or a Jasco UV-2070 Plus Intelligent UV/VIS Detector at 278 nm (Jasco).