Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris.

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35-37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg2+, Mn2+, Li+, Ca2+, Ni2+, CHAPS, DTT, and EDTA and inhibited by Hg2+, Ag+, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.

The potential of cellulosic ethanol production from grasses in Thailand.

The grasses in Thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process. The average percentage composition of cellulose, hemicellulose, and lignin in the samples of 18 types of grasses from various provinces was determined as 31.85-38.51, 31.13-42.61, and 3.10-5.64, respectively. The samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification. The total reducing sugars in most grasses ranging from 500-600 mg/g grasses (70-80% yield) were obtained. Subsequently, 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation (SSCF). The enzymes, cellulase and xylanase, were utilized for hydrolysis and the yeasts, Saccharomyces cerevisiae and Pichia stipitis, were applied for cofermentation at 35 °C for 7 days. From the results, the highest yield of ethanol, 1.14 g/L or 0.14 g/g substrate equivalent to 32.72% of the theoretical values was obtained from Sri Lanka ecotype vetiver grass. When the yields of dry matter were included in the calculations, Sri Lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 L/ha/year, whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 L/ha/year (0.98 g/L or 0.12 g/g substrate equivalent to 30.60% of the theoretical values).