Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Effect of Hyperbaric Oxygen Therapy (HBOT) on Insulin Resistance Associated with Abdominal Obesity in Wistar Rats with Dietary Sucrose-Induced Metabolic Syndrome.

Metabolic syndrome (MS) is a combination of risk factors related to the development of mainly type 2 diabetes mellitus, cardiovascular disease (CVD) and nonalcoholic fatty liver disease (NAFLD). Its prevalence has increased worldwide, and healthcare systems will face major challenges in addressing this problem. The aim of this work was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on insulin resistance (IR) and obesity associated with MS in Wistar rats. The experimental design consisted of three groups of sucrose-induced MS rats: the MS group that consumed sucrose (MS-Suc; n=5), the MS group that ingested sucrose and HBOT (MS-Suc-HBOT; n=5), the MS group that did not consume sucrose and that received HBOT (MS-HBOT; n=5) and the control group. The rats received HBOT for 20 d at 2.4 atmospheres absolute (ATA) for 60 min. Subsequently, the rats were euthanized, and body fat weight, serum biochemical parameters and microscopic analysis of adipose tissue were determined. Rats with hyperoxia had decreased body weight, adipose tissue hypertrophy, and abdominal and epididymal fat. Likewise, markers of insulin resistance (glucose, insulin and HOMA-IR), biochemical parameters of dyslipidemia (cholesterol and triglycerides) and nonalcoholic fatty liver (AST and ALT) decreased; in contrast, compared to the control group, HBOT increased the 1/HOMA-IR, HOMA-ßCell and McAuley indexes, which were related to the improvement in insulin sensitivity (p<0.05; p<0.01). HBOT showed beneficial effects in the treatment of IR and obesity associated with sucrose-induced metabolic syndrome in Wistar rats.

Curcumin Nanoemulsions Stabilized with Modified Phosphatidylcholine on Skin Carcinogenesis Protocol.

BACKGROUND: Cancer is one of the main causes of death by disease; several alternative treatments have been developed to counteract this condition. Curcumin (diferuloylmethane), extracted from the rhizome of Curcuma longa, has antioxidant, anti-inflammatory, and anti-cancer properties; however, it has low water solubility and poor intestinal absorption. Carrier systems, such as nanoemulsions, can increase the bioavailability of lipophilic bioactive compounds. OBJECTIVE: To evaluate the effect of curcumin nanoemulsions prepared with lecithin modified with medium-chain fatty acids as an emulsifier, on the expression of the Cdk4, Ccne2, Casp8 and Cldn4 genes involved in the carcinogenesis process in K14E6 transgenic mice. METHODS: The emulsifier was prepared by interesterification of medium-chain fatty acids, pure lecithin, and immobilized phospholipase-1 on Duolite A568. An Ultraturrax homogenizer and a Branson Ultrasonic processor were used for the preparation of nano-emulsions, and a Zetasizer evaluated the particle size. qRT-PCR analysis was performed to quantify the cancer-related genes expressed in the K14E6 mice. The development and evolution of skin carcinogenesis were assessed through histological analysis to compare cell morphology. RESULTS: Ca 59% of the MCFA were incorporated via esterification into the PC within 12 hours of the reaction. An emulsifier yield used to formulate the NE of 86% was achieved. Nanoemulsions with a particle size of 44 nm were obtained. The curcumin nano-emulsion group had a 91.81% decrease in the tumorigenesis index and a reduction in tumor area of 89.95% compared to the sick group. Histological analysis showed that the group administered with free curcumin developed a microinvasive squamous cell carcinoma, as opposed to the group with nanoemulsion which presented only a slight inflammation. In gene expression, only a significant difference in Cdk4 was observed in the nanoemulsion group.

Comparative Survival and the Cold-Induced Gene Expression of Pathogenic and Nonpathogenic Vibrio Parahaemolyticus from Tropical Eastern Oysters during Cold Storage.

Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by -0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.

Cd36 gene expression in adipose and hepatic tissue mediates the lipids accumulation in liver of obese rats with sucrose-induced hepatic steatosis.

Obesity is considered a global epidemic and is mainly associated with the development of diabetes, cardiovascular diseases and Non-Alcoholic Fatty Liver (NAFLD). The pathogenesis between obesity and hepatic steatosis is partially known, but could involve differentiated or tissue-specific participation of the expression of Cd36 mRNA that codes for a receptor which is a transporter of free fatty acids (FFA) in different tissues, favoring the lipids storage. This relative expression was evaluated in adipose and liver tissue in rats with steatosis after consumption of sucrose for 30 and 40 weeks. Ten Wistar rats were divided into two experimental groups (St-30 and St-40), which received a standard diet plus 30 % sucrose in their water intake. These rats showed a significant increase in abdominal fat, serum biochemical determinations, HOMA-IR; as well as, changes in adipocytes size and mild portal hepatitis and grade 2 hepatic steatosis. The relative expression of Cd36 mRNA increased in liver tissue after 30 (4.5-fold) and 40 (8.5-fold) weeks of sucrose ingestión but no in adipose tissue; with respect to control group (P < 0.05). This expression was associated with a significant increase in the levles of sCD36 in serum, which is indicator of the presence of the FFA transporter in the hepatocyte membrane causing lipids accumulation. The above shows the link between the adipose and hepatic tissue for the accumulation of steatotic fat in the liver through time, mediated by the relative expression of cd36 mRNA that encodes for the FFA transporter.

Gene Cloning and Characterization of the Geobacillus thermoleovorans CCR11 Carboxylesterase CaesCCR11, a New Member of Family XV.

A gene encoding a carboxylesterase produced by Geobacillus thermoleovoras CCR11 was cloned in the pET-3b cloning vector, sequenced and expressed in Escherichia coli BL21(DE3). Gene sequence analysis revealed an open reading frame of 750 bp that encodes a polypeptide of 250 amino acid residues (27.3 kDa) named CaesCCR11. The enzyme showed its maximum activity at 50 °C and pH 5-8, with preference for C4 substrates, confirming its esterase nature. It displayed good resistance to temperature, pH, and the presence of organic solvents and detergents, that makes this enzyme biotechnologically applicable in the industries such as fine and oleo-chemicals, cosmetics, pharmaceuticals, organic synthesis, biodiesel production, detergents, and food industries. A 3D model of CaesCCR11 was predicted using the Bacillus sp. monoacyl glycerol lipase bMGL H-257 structure as template (PBD code 3RM3, 99 % residue identity with CaesCCR11). Based on its canonical α/ß hydrolase fold composed of 7 ß-strands and 6 α-helices, the α/ß architecture of the cap domain, the GLSTG pentapeptide, and the formation of distinctive salt bridges, we are proposing CaesCCR11 as a new member of family XV of lipolytic enzymes.

High hydrostatic pressure induces synthesis of heat-shock proteins and trehalose-6-phosphate synthase in Anastrepha ludens larvae.

The Mexican fruit fly (Anastrepha ludens) is responsible for losses of up to 25% of crops such as mango and citrus fruits in Central America and México. The larval life cycle of A. ludens comprises three stages with a duration ranging from 3 to 8 days. Because of the damage caused by A. ludens, several methods of control have been studied and implemented. High hydrostatic pressures (HHP) are currently applied to foods and it is now proposed to be employed to inactivate eggs and larvae of A. ludens. Originally HHP was designed to inactivate microorganisms, since it exerts marked effects on cell morphology, and can affect enzymatic reactions and genetic mechanisms of microbial cells, with no major changes altering the sensory or nutritional quality of the foodstuff. In this study, A. ludens in two larval stages (5- and 8-day-old) were subjected to HHP treatments. The biochemical response of the larvae of A. ludens was dependent on their stage of development. The third larval stage (L3) developed a better protection mechanism based on the synthesis of stress proteins or heat-shock proteins (HSPs) and the enzyme trehalose-6-phosphate synthase, which are linked and possibly act together to achieve greater survivability to stress caused by hydrostatic pressure.

Enzymatic reactions and synthesis of n-butyl caproate: esterification, transesterification and aminolysis using a recombinant lipase from Geobacillus thermoleovorans CCR11.

The recombinant lipase LipMatCCR11 from the thermophilic strain Geobacillus thermoleovorans CCR11 was applied in the synthesis of n-butyl caproate via transesterification in hexane and xylene. The short chain flavour ester was obtained by alcoholysis from ethyl caproate and n-butyl alcohol and acidolysis from n-butyl butyrate and caproic acid. This enzyme was also used in the condensation reaction from caproic acid and n-butanol. The conversion percentages at equilibrium (Xe) were similar to those obtained with Candida antarctica lipase fraction B (CAL-B) in the same reaction conditions, while lower conversion velocities (k) were attained. LipMatCCR11 reached high conversion percentages in either hexane or xylene as organic media (> 63%); the enzyme was also able to catalyze the aminolysis reaction of ethyl caproate with benzyl amine in hexane obtaining a conversion percentage > 62%.

Gene cloning, expression, and characterization of the Geobacillus Thermoleovorans CCR11 thermoalkaliphilic lipase.

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.

Expression of the cry11A gene of Bacillus thuringiensis ssp. israelensis in Saccharomyces cerevisiae.

The complete cry11A region gene of Bacillus thuringiensis ssp. israelensis was fused in frame to the 3' end of the GST gene under the control of the Saccharomyces cerevisiae HXK1 promoter. The fusion protein GST-cry11A was expressed in S. cerevisiae strain AMW13C+. The fusion gene GST-cry11A was expressed when yeast cells were grown on galactose and a nonfermentable medium containing ethanol as carbon and energy source. When the cells were grown in glucose, mannose, fructose, or glycerol as carbon sources, the GST-cry11A gene was repressed. Thus, a regulated expression in accordance with the regulatory activity of the HXK1 gene promoter has been detected. The GST-cry11A fusion protein was detected in the transformed yeasts as a soluble protein. The fusion protein was purified by affinity chromatography using glutathione-Sepharose beads. Cell-free extracts from transformed yeasts grown in ethanol-containing culture media showed insecticidal activity against third-instar Aedes aegypti larvae. This insecticidal activity was increased about 4-fold when the purified fusion protein was assayed.