María Eugenia Hidalgo-Lara

Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cfl xyn11A) from Cellulomonas flavigena

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse.

Synergy between Cellulomonas flavigena xylanase CflXyn11A and Trichoderma reesei endoglucanase TrCel7B was assessed during hydrolysis of alkaline pretreated sugar cane bagasse (SCB) after 12-48 h, applying the individual enzymes and mixtures of the enzymes. A high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of SCB was observed after 12h in the equimolar mixture. A threefold decrease in the degree of synergy was observed with TrCel7B and the catalytic module of CflXyn11A; suggesting an important role played by the carbohydrate-binding module of CflXyn11A (CflXyn11A-CBM) in the observed synergy. Affinity electrophoresis and binding assays showed that CflXyn11A-CBM binds to xylans and to a lesser extent to cellulose. Our results suggest that synergy is more pronounced at early stages of hydrolysis. Furthermore, for the first time it is described that a CBM carried by a xylanase significantly enhances the synergy with a cellulase (threefold increase in synergy).

Immobilization of Recombinant Invertase (re-INVB) from Zymomonas mobilis on D-Sorbitol Cinnamic Ester for Production of Invert Sugar

The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized enzyme was 5, and the optimal reaction temperature was 40 degrees C. The apparent Michaelis constant and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose was 78+/-5 mM and 5x10(4)+/-3x10(2) s(-1), respectively, while for the free enzyme, it was 98.0+/-4 mM and 1.2x10(4)+/-2.5x10(2) s(-1), respectively, suggesting a better apparent affinity of the enzyme for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized re-INVB also showed good thermal stability and good operational stability (40% of the initial activity remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per 2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol are an appropriate support for re-INVB immobilization and the production of invert sugar.

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.

Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.