Isolation, overexpression and characterization of an alkaline˗stable lipase KV1 from acinetobacter haemolyticus

The study reports the purification and comprehensive biochemical characterization of a novel lipase KV1 (LipKV1) from Acinetobacter haemolyticus strain KV1. Strain KV1 was identified as Acinetobacter haemolyticus based on results of 16S rDNA sequencing, phylogenetic and BIOLOG. The intracellular wild-type LipKV1 was purified to offer specific activity of 32 U/mg with an estimated relative molecular mass of 37 kDa. The PCR product of LipKV1 revealed that the retrieved sequence contained the proposed complete lipase gene sequence at nucleic acid positions 1~954. The purified wild-type LipKV1 exhibited a maximum relative activity at 40°C and pH 8.0. The lipase was activated (112-128%) in Na+, Ca2+, K+ and Mg2+ and the enzyme hydrolyzed a wide range of oils with tributyrin (140%) being the preferred ones. Reducing (PMSF, DTT, β-mercaptoethanol) and chelating (EDTA) agents significantly inhibited the LipKV1 relative activity (p < 0.05). Surfactants Tween 20-80 (110-143%) significantly enhanced the relative activity (p < 0.05). Gene encoding intracellular lipase was cloned to produce a large quantity of the recombinant LipKV1. The lipase which contained His-tag was expressed in Esherichia coli BL21 (DE3) cells using pET-30a as expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG concentration, post-induction temperature and post-induction time) were performed. All parameters were significant (p < 0.05) in influencing the expression of LipKV1, rendering a 70% increase in enzyme production at optimum induction conditions. The expressed recombinant LipKV1 was purified using Ni-affinity chromatography, to a specific activity of 233.4 U/mg and an estimated relative molecular mass of 39 kDa. The recombinant LipKV1 exhibited a maximum activity at 40°C and pH 8.0. Homology modeling of the lipase structure was carried out based on the template structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus, which shares a 58% sequence identity to LipKV1. The LipKV1 model comprised a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three conserved active-site residues, namely Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the active site of LipKV1 modeled at pH 8.0 revealed an aromatic platform responsible for the substrate recognition and preference towards tributyrin. The binding modes from the docking simulation appear to correlate well with the experimentally determined hydrolysis pattern, for which pH 8.0 is optimum and tributyrin being the preferred substrate. A low Km value (0.6 mM) for tributyrin further verifies the high affinity of LipKV1 for the substrate. Biophysical characterization of recombinant LipKV1 protein using ultaviolet-visible (UV-Vis) spectroscopy, circular dichroism (CD), fluorescence spectroscopy, ANS fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) indicated that the lipase retains its secondary structure and good folding at alkaline pH conditions (pH 8.0 and pH 12.0) and at 40C. Alkaline-stable enzymes such as LipKV1 are therefore, useful in biotechnology-based industries in order to shorten production time, minimizing energy consumption and preventing undesired chemical transformations

Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

Background. Oxidative stress induced by reactive oxygen and nitrogen species is critically involved in the impairment of β-cell function during the development of diabetes. Methods. HIT-T15 cells were cultured in 5% CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 µg/mL) as well as quercetin (20, 40, 60, and 80 µM), prior to stimulation by 20 and 50 mM of glucose. Cell lysate was collected to determine the effect of honey extracts and quercetin on the stress activated NF-κB, MAPK pathways, and the Akt (ser473) activated insulin signaling pathway. Results. HIT-T15 cells cultured under hyperglycemic conditions demonstrated insulin resistance with a significant increase in the levels of MAPK, NF-κB, and IRS-1 serine phosphorylation (ser307); however, Akt expression and insulin contents are significantly decreased. Pretreatment with quercetin and Gelam honey extract improved insulin resistance and insulin content by reducing the expression of MAPK, NF-κB, and IRS-1 serine phosphorylation (ser307) and increasing the expression of Akt significantly. Conclusion. Gelam honey-induced differential expression of MAPK, NF-κB, IRS-1 (ser307), and Akt in HIT-T15 cells shows that Gelam honey exerts protective effects against diabetes- and hyperglycemia-induced oxidative stress by improving insulin content and insulin resistance

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in umbilical vein endothelial cells

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (

Molecular Targets of Aptamers in Gastrointestinal Cancers: Cancer Detection, Therapeutic Applications, and Associated Mechanisms

Gastrointestinal (GI) cancers are among the most common cancers that impact the global population, with high mortality and low survival rates after breast and lung cancers. Identifying useful molecular targets in GI cancers are crucial for improving diagnosis, prognosis, and treatment outcomes, however, limited by poor targeting and drug delivery system. Aptamers are often utilized in the field of biomarkers identification, targeting, and as a drug/inhibitor delivery cargo. Their natural and chemically modifiable binding capability, high affinity, and specificity are favored over antibodies and potential early diagnostic imaging and drug delivery applications. Studies have demonstrated the use of different aptamers as drug delivery agents and early molecular diagnostic and detection probes for treating cancers. This review aims to first describe aptamers’ generation, characteristics, and classifications, also providing insights into their recent applications in the diagnosis and medical imaging, prognosis, and anticancer drug delivery system of GI cancers. Besides, it mainly discussed the relevant molecular targets and associated molecular mechanisms involved, as well as their applications for potential treatments for GI cancers. In addition, the current applications of aptamers in a clinical setting to treat GI cancers are deciphered. In conclusion, aptamers are multifunctional molecules that could be effectively used as an anticancer agent or drug delivery system for treating GI cancers and deserve further investigations for clinical applications

Matrix metalloproteinases in chemoresistance: regulatory roles, molecular interactions, and potential inhibitors

Cancer is one of the major causes of death worldwide. Its treatments usually fail when the tumor has become malignant and metastasized. Metastasis is a key source of cancer recurrence, which often leads to resistance towards chemotherapeutic agents. Hence, most cancer-related deaths are linked to the occurrence of chemoresistance. Although chemoresistance can emerge through a multitude of mechanisms, chemoresistance and metastasis share a similar pathway, which is an epithelial-to-mesenchymal transition (EMT). Matrix metalloproteinases (MMPs), a class of zinc and calcium-chelated enzymes, are found to be key players in driving cancer migration and metastasis through EMT induction. The aim of this review is to discuss the regulatory roles and associated molecular mechanisms of specific MMPs in regulating chemoresistance, particularly EMT initiation and resistance to apoptosis. A brief presentation on their potential diagnostic and prognostic values was also deciphered. It also aimed to describe existing MMP inhibitors and the potential of utilizing other strategies to inhibit MMPs to reduce chemoresistance, such as upstream inhibition of MMP expressions and MMP-responsive nanomaterials to deliver drugs as well as epigenetic regulations. Hence, manipulation of MMP expression can be a powerful tool to aid in treating patients with chemo-resistant cancers. However, much still needs to be done to bring the solution from bench to bedside

Biochemical and in-silico structural assessments of an acinetobacter haemolyticus lipase KV1 isolated from an oil palm mill effluent

The use of microbial enzymes as biocatalysts for a myriad of commercial processes are currently trending owing to their versatility and, their use is considerably greener than the chemically-assisted methods. In this regard, this study reports the comprehensive biochemical characterization of a lipase from novel Acinetobacter haemolyticus KV1 bacteria. The intracellular lipase was purified to ~3.5-fold using consecutive treatments of ammonium sulfate precipitation, dialysis and DEAE-cellulose ion exchange chromatography. The purified lipase exhibited maximum relative activity at 40°C and pH 8.0, respectively. Lipase KV1 was significantly activated (p 50% for up to 24 h for pH between pH 7-11. Therefore, the full characterization of lipase KV1 reported in this study deserves scientific and economic considerations

Biophysical characterization of a recombinant lipase KV1 from Acinetobacter haemolyticus in relation to pH and temperature

Spectroscopic and calorimetric methods were employed to assess the stability and the folding aspect of a novel recombinant alkaline-stable lipase KV1 from Acinetobacter haemolyticus under varying pH and temperature. Data on far ultraviolet-circular dichroism of recombinant lipase KV1 under two alkaline conditions (pH 8.0 and 12.0) at 40 °C reveal strong negative ellipticities at 208, 217, 222 nm, implying its secondary structure belonging to a α + β class with 47.3 and 39.0% ellipticity, respectively. Results demonstrate that lipase KV1 adopts its most stable conformation at pH 8.0 and 40 °C. Conversely, the protein assumes a random coil structure at pH 4.0 and 80 °C, evident from a strong negative peak at ∼ 200 nm. This blue shift suggests a general decline in enzyme activity in conjunction with the partially or fully unfolded state that invariably exposed more hydrophobic surfaces of the lipase protein. The maximum emission at ∼335 nm for pH 8.0 and 40 °C indicates the adoption of a favorable protein conformation with a high number of buried tryptophan residues, reducing solvent exposure. Appearance of an intense Amide I absorption band at pH 8.0 corroborates an intact secondary structure. A lower enthalpy value for pH 4.0 over pH 8.0 and 12.0 in the differential scanning calorimetric data corroborates the stability of the lipase at alkaline conditions, while a low K m (0.68 ± 0.03 mM) for tributyrin verifies the high affinity of lipase KV1 for the substrate. The data, herein offer useful insights into future structure-based tunable catalytic activity of lipase KV1

The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions

Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic β-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 μg/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 μM), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the β-cells from hamsters and its cytoprotective effect against hyperglycemia

A statistical approach for optimizing the protocol for overexpressing lipase KV1 in Escherichia coli: purification and characterization

Lipase is one of the most important industrial enzymes, widely used in the preparation of food additives, cosmetics and pharmaceuticals. In order to obtain a large amount of lipase, in the present study, a gene encoding intracellular lipase was cloned from Acinetobacter haemolyticus. The recombinant lipase KV1 containing a His-tag was expressed in Esherichia coli BL21 (DE3) cells, using pET-30a as the expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, post-induction temperature and post-induction time) were made. All parameters significantly (P 80%) even up to 24 h between pH 7−12; suggesting that the recombinant lipase KV1 may be suitable for a wide range of industrial applications

Molecular Mechanisms of Diabetic Retinopathy, General Preventive Strategies, and Novel Therapeutic Targets

The growing number of people with diabetes worldwide suggests that diabetic retinopathy (DR) and diabetic macular edema (DME) will continue to be sight threatening factors. The pathogenesis of diabetic retinopathy is a widespread cause of visual impairment in the world and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. Despite understanding the polyol pathway flux, activation of protein kinase C (KPC) isoforms, increased hexosamine pathway flux, and increased advanced glycation end-product (AGE) formation, pathogenic mechanisms underlying diabetes induced vision loss are not fully understood. The purpose of this paper is to review molecular mechanisms that regulate cell survival and apoptosis of retinal cells and discuss new and exciting therapeutic targets with comparison to the old and inefficient preventive strategies. This review highlights the recent advancements in understanding hyperglycemia-induced biochemical and molecular alterations, systemic metabolic factors, and aberrant activation of signaling cascades that ultimately lead to activation of a number of transcription factors causing functional and structural damage to retinal cells. It also reviews the established interventions and emerging molecular targets to avert diabetic retinopathy and its associated risk factors