Kinetics and thermodynamics of thermal inactivation for recombinant Escherichia coli cellulases, cel12B, cel8C, and polygalacturonase, peh28; biocatalysts for biofuel precursor production.

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in Escherichia coli and their potential in biofuel production.

BACKGROUND: The high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of celB-, celC-encoded cellulases (cel12B and cel8C) and peh-encoded polygalacturonase (peh28) from Pectobacterium carotovorum subsp. carotovorum (Pcc) was carried out in our previous study using Escherichia coli as a host vector. The current study partially characterizes the enzymes' molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion. RESULTS: ß-Jelly roll topology, (α/α)6 antiparallel helices and right-handed ß-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed ß-glucosidase activities were identified for cel8C and cel12B when tested on p-nitrophenyl-ß-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant ß-glucosidase from Pcc was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (kcat/Km) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and ß-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin. CONCLUSIONS: This study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal.

The effect of leptin on luteal angiogenic factors during the luteal phase of the estrous cycle in goats.

Fibroblast growth factor 2 (FGF2), angiopoietin 1 (Ang1), and vascular endothelial growth factor (VEGF) are angiogenic factors implicated in the vascular development of the corpus luteum (CL). Each factor is regulated or influenced by leptin in non-ovarian tissues. Moreover, leptin and its receptor, ObRb, have been identified in luteal tissue throughout the luteal phase. Therefore, leptin is hypothesized to influence luteal vasculature through the regulation of FGF2, Ang1, and VEGF. Multiparous, cycling crossbred female goats (does) were allocated to early (n=12), mid (n=8), and late (n=11) stages of the luteal phase for CL collection. Luteal tissue was harvested and either snap frozen in liquid N2, paraffin embedded, or cultured with leptin (0, 10(-12), 10(-11), 10(-10), 10(-9), 10(-8)M). Tissue was analyzed for FGF2, Ang1, VEGF, ObRb, and leptin expression. Angiopoietin 1, FGF2, VEGF expression was higher (P≤0.001) in the mid-luteal stage than the early stage. Expression decreased (P≤0.001) during the late luteal stage with the exception of VEGF, which remained elevated. In contrast, leptin and ObRb were lowest (P≤0.003) during the mid-luteal stage compared to the early and late stages. All factors were detected in and/or around vessels in early stage tissue compared to mid and late stages. Leptin stimulated (P≤0.02) Ang1, FGF2, and VEGF expression only in early stage luteal cultures. Collectively, these data provide evidence that leptin may be involved in the luteal angiogenic process during the early stage of CL formation.

Microarray analysis of Foxl2 mediated gene regulation in the mouse ovary derived KK1 granulosa cell line: Over-expression of Foxl2 leads to activation of the gonadotropin releasing hormone receptor gene promoter.

BACKGROUND: The Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary. METHODS: Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software. RESULTS: Microarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an activator of the GnRHR gene. CONCLUSIONS: Potential Foxl2 regulated ovarian genes have been identified through microarray analysis and comparison of these data to other microarray studies. The Foxl2 responsiveness of the GnRHR gene has been validated and provided evidence of Foxl2 transcriptional activation of the GnRHR gene promoter in the mouse ovary derived KK1 granulosa cell line.