RESUMO
BACKGROUND: Cellulose is the most important component of lignocellulose, and its degradation requires three different types of enzymes to act synergistically. There have been reports of single gene duality, but no gene has been described to have more than two functions. Cloning and expression of fusion cellulases containing more than two kinds of catalytic domains has not been reported thus far. RESULTS: We synthesized three different cellulase genes and linked the three catalytic domains with a (G4S)3 flexible linker. The trifunctional cellulase gene (BCE) containing three types of cellulase functions was constructed and expressed in S. cerevisiae successfully. The ß-glucosidase, the exoglucanase and the endoglucanase activity of the trifunctional cellulase BCE reached 16.80 IU/mg, 2.26 IU/mg and 20.67 IU/mg, which was 46.27, 6.73 and 46.20% higher than the activities of the ß-glucosidase BG, the endoglucanase CBH and the endoglucanase EG. The filter paper enzyme activity of BCE was higher than those of BG, CBH and EG, reached 2.04 IU/mg. CONCLUSIONS: The trifunctional cellulase BCE was designed based on ß-glucosidase BG, endoglucanase EG and exoglucanase CBH, and it possessed ß-glucosidase activity, endoglucanase activity and exoglucanase activity simultaneously. The BCE has better filter paper activity, it means the potential practical application.
Assuntos
Celulase , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae , beta-Glucosidase , Domínio Catalítico , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Lignina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismoRESUMO
Lignocellulose is considered as a good resource for producing renewable energy. Previous in vitro studies have shown the synergistic action between cellulase and xylanase during lignocellulose biohydrolysis. In order to achieve the same effect in S. cerevisiae to enhance the practical biotransformation, two recombinant Saccharomyces cerevisiae strains (INVSc1-CBH-CA and INVSc1-CBH-TS) with co-expressed cellulase and xylanase were constructed. The cellulase and xylanase activities in INVSc1-CBH-CA and INVSc1-CBH-TS were 716.43 U/mL and 205.13 U/mL, 931.27 U/mL and 413.70 U/mL, respectively. The recombinant S. cerevisiae can use the partly delignified corn stover (PDCS) more efficiently and more ethanol producted than S. cerevisiae only expressing cellulase. Fermentation with INVSc1-CBH-CA and INVSc1-CBH-TS using PDCS ethanol yields increased by 1.7 and 2.1 folds higher than INVSc1-CBH, 2.8 and 3.4 folds higher than the wild type S. cerevisiae. The strategy of co-expression cellulase and xylanase in saccharomyces cerevisiae is effective and can be a foundation to research the mechanism of synergy effect of cellulose and xylanase.
Assuntos
Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Zea mays/microbiologia , Celulase/genética , Endo-1,4-beta-Xilanases/genética , Fermentação , Expressão Gênica , Lignina/metabolismo , Saccharomyces cerevisiae/genética , Resíduos/análise , Zea mays/química , Zea mays/metabolismoRESUMO
Lignocellulose is a polysaccharide and an abundant biomass resource that widely exists in grains, beans, rice, and their by-products. Over 10 million tons of lignocellulose resources and processing products are produced every year in China. Three recombinant Y. lipolytica strains with cellulase (ß-glucosidase, endoglucanase and cellobiohydrolase) were constructed. The enzymatic activities of these enzymes were 14.181 U/mL, 16.307 U/mL, and 17.391 U/mL, respectively. The whole cell cellulases were used for a stover bio-transformation. The celluloses in the stover were partly degraded by the cellulases, and the degradation products were transformed into single cell protein (SCP) by the Y. lipolytica cells. After 15 d of fermentation with the whole cell cellulases, the protein content of the maize stover and the rice straw reached 16.23% and 14.75%, which increased by 168.26% and 161.52% compared with the control, respectively. This study provides a new stage for the efficient utilization of stover in the feed industry.
Assuntos
Celulases/genética , Lignina/metabolismo , Engenharia Metabólica/métodos , Oryza/microbiologia , Recombinação Genética/genética , Yarrowia/fisiologia , Biotransformação/genética , Melhoramento Genético/métodos , Componentes Aéreos da Planta/microbiologiaRESUMO
Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase.
Assuntos
Celulase/química , Lignina/química , Oryza/química , Xilosidases/química , Zea mays/química , Biotecnologia/métodos , Carboidratos/química , Celulase/metabolismo , Celulose/química , Celulose/metabolismo , Hidrólise , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Oryza/metabolismo , Brotos de Planta/química , Brotos de Planta/metabolismo , Xilosidases/metabolismo , Zea mays/metabolismoRESUMO
Cellulose is an abundant natural polysaccharide that is universally distributed. It can be extracted from corncobs, which are inexpensive, easily accessible, renewable, and environmentally friendly. A common strategy for effectively utilizing cellulose is efficient heterogeneous expression of cellulase genes in Saccharomyces cerevisiae. However, the improvement of cellulose utilization is a relevant issue. Based on our previous findings, we constructed an integrated secretion expression vector, pHBM368-pgk, containing a constitutive promoter sequence. Three genetically modified S. cerevisiae strains containing heterologous ß-glucosidase, exoglucanase, and endoglucanase genes were constructed. The results of a 1-L bioreactor fermentation process revealed that the mixed recombinant S. cerevisiae could efficiently carry out simultaneous saccharification and fermentation (SSF) by using corncobs as the sole carbon source. The ethanol concentration reached 6.37 g/L after 96 hours of fermentation, which was about 3 times higher than that produced by genetically modified S. cerevisiae with the inducible promoter sequence. To investigate the microstructure characteristics of hydrolyzed corncobs during the fermentation process, corncob residues were detected by using a scanning electron microscope. This study provides a feasible method to improve the effect of SSF using corncobs as the sole carbon source.