Two novel lipases purified from rice bran displaying lipolytic and esterification activities.

In this study, two novel lipases, rice bran lipase 1 (RBL1) and rice bran lipase 2 (RBL2), were first identified in rice (Oryza sativa) bran. Through the purification by ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography, RBL1 and RBL2 were purified to 36- and 339-fold with the final specific activity of 4.73 and 44.06 U/mg, respectively. The purified RBL1 and RBL2 had the molecular weight of 18.8 and 35.5 kDa, respectively. The Km values of RBL1 and RBL2 were 0.766 and 0.432 mM while catalytic efficiency (kcat/Km) values of RBL1 and RBL2 were 102.4 and 2559.3 s-1/mM, respectively. RBL1 and RBL2 both showed esterification activity, but had no transesterification activity. In a solvent-free system, RBL1 and RBL2 exhibited degree of esterification (ED) of 4.4% and 6.5%, respectively. These two novel lipases exerted great properties for their potentials in industrial applications. First, RBL1 and RBL2 showed both mild reaction pH of 7.0 and temperature of 35 °C and 50 °C, respectively. Secondly, they showed great tolerance to several organic solvents and detergents while RBL1 exhibited great pH stability across a very broad range of pH (pH 3-12). Lastly and most importantly, RBL1 and RBL2 both exhibited esterification activity better than a commercial lipase, Candida rugosa lipase (CRL), in a solvent-free system. In conclusion, two novel lipases, RBL1 and RBL2, are different from published native lipases in rice bran and may be alternative potential candidates of biocatalysts contributing to the development of diverse industrial application fields.

Bi-functional fusion enzyme EG-M-Xyn displaying endoglucanase and xylanase activities and its utility in improving lignocellulose degradation.

In this study, the gene fusion of endoglucanase (EG, one of cellulases) from Teleogryllus emma and xylanase (Xyn, one of hemicellulases) from Thermomyces lanuginosus was constructed to generate a fusion enzyme (EG-M-Xyn). Through the expression and purification by ultrafiltration and size-exclusion chromatography, the purified EG-M-Xyn had a molecular weight of 75.5 kDa and exhibited the specific activity of CMCase and xylanase as 306.8 U/mg and 1227.3 U/mg, respectively. The Km values (CMC and beechwood xylan) were 6.8 and 60.6 mg mL-1 while catalytic efficiency (kcat/Km) values of CMCase and xylanase were 3280 and 38,797 min-1 mg-1 mL, respectively. EG-M-Xyn exerted great properties for its great potential in improving the enzymatic hydrolysis of lignocellulosics to produce fermentable sugars. First, EG-M-Xyn showed mild reaction pH and temperature of 5.5 and 50 °C, respectively. Secondly, EG-M-Xyn exhibited great heat tolerance of T1/2 values of 173 (CMCase) and 693 min (xylanase). Lastly and most importantly, application of EG-M-Xyn in combination with Ctec2 (commercial enzyme) in the saccharification led to a 10-20% net increase in fermentable sugars liberated from pretreated rice straw in comparison to the Ctec2 alone group. In conclusion, EG-M-Xyn had great potential in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industry.

Expression of Synthetic Phytoene Synthase Gene to Enhance ß-Carotene Production in Scenedesmus sp. CPC2.

ß-carotene is a valuable pigment abundant in some microalgal species but the low ß-carotene productivity of microalgae has become the major obstacles against its commercialization. This work aims to improve the productivity of algae-based ß-carotene via genetic engineering approaches. First, a synthetic psy gene construct (891 bp) encoding 297 amino acids is expressed in Scenedesmus sp. CPC2 host to enhance the ß-carotene production. The synthetic psy gene is designed by considering the highest consensus of amino acids (i.e., 62% identity) from Chlamydomonas reinhardtii, Dunaliella salina, and Mariella zofingiensis. The original ß-carotene content in wild-type Scenedesmus sp. CPC2 is 10.8 mg g-1 -cell when grown on BG11 medium under 2% CO2 aeration, 150 µmol m-2 s-1 light intensity and 25°C. After transformation of the psy gene into the microalgal host, the ß-carotene content of the best recombinant strain (i.e., transformant CPC2-4) significantly increased to over 30 mg g-1 -cell. The optimal production of ß-carotene with the CPC2-4 recombinant strain was achieved when the strain is grown on BG11 medium amended with 0.075 g of MgSO4 , giving approximately 3-fold higher ß-carotene content than that of the wild-type strain. The best cellular ß-carotene content obtained (i.e., 31.8 mg g-1 ) is superior to most algae-based ß-carotene production performance reported in the literature.

Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production.