Madiha Bou Ali

Hypoglycemic and antilipidemic properties of kombucha tea in alloxan-induced diabetic rats.

BackgroundDiabetes has become a serious health problem and a major risk factor associated with troublesome health complications, such as metabolism disorders and liver-kidney dysfunctions. The inadequacies associated with conventional medicines have led to a determined search for alternative natural therapeutic agents. The present study aimed to investigate and compare the hypoglycemic and antilipidemic effects of kombucha and black tea, two natural drinks commonly consumed around the world, in surviving diabetic rats.MethodsAlloxan diabetic rats were orally supplied with kombucha and black tea at a dose of 5 mL/kg body weight per day for 30 days, fasted overnight, and sacrificed on the 31st day of the experiment. Their bloods were collected and submitted to various biochemical measurements, including blood glucose, cholesterol, triglcerides, urea, creatinine, transaminases, transpeptidase, lipase, and amylase activities. Their pancreases were isolated and processed to measure lipase and α-amylase activities and to perform histological analysis.ResultsThe findings revealed that, compared to black tea, kombucha tea was a better inhibitor of α-amylase and lipase activities in the plasma and pancreas and a better suppressor of increased blood glucose levels. Interestingly, kombucha was noted to induce a marked delay in the absorption of LDL-cholesterol and triglycerides and a significant increase in HDL-cholesterol. Histological analyses also showed that it exerted an ameliorative action on the pancreases and efficiently protected the liver-kidney functions of diabetic rats, evidenced by significant decreases in aspartate transaminase, alanine transaminase, and gamma-glytamyl transpeptidase activities in the plasma, as well as in the creatinine and urea contents.ConclusionsThe findings revealed that kombucha tea administration induced attractive curative effects on diabetic rats, particularly in terms of liver-kidney functions. Kombucha tea can, therefore, be considered as a potential strong candidate for future application as a functional supplement for the treatment and prevention of diabetes.

Purification and biochemical characterization of a halotolerant Staphylococcus sp. extracellular lipase

We have isolated a lipolytic halotolerant bacterium, designated as CJ3, that was identified as a Staphylococcus sp. Culture conditions were optimized and the highest extracellular lipase production amounting to 5 U/ml was achieved after 24 h of cultivation. The extracellular lipase was purified 24-fold by ammonium sulfate precipitation and a Sephacryl S-200 chromatography, and its molecular mass was found to be around 38 kDa, as revealed by SDS-PAGE and gel filtration. The lipase substrate specificity was investigated using short (tributyrin) and long (olive oil) chain triglyceride substrates. The lipase was inhibited by submicellar concentrations of Triton X-100, and maximum specific activities were found to be 802 U/mg on tributyrin and 260 U/mg on olive oil at pH 8.0 and 45 °C. The lipase was fairly stable in the pH range from 6.0 to 9.0, and about 69% of its activity was retained after incubation at 45 °C for 60 min. The enzyme showed a high tolerance to a wide range of salt concentration and a good stability in organic solvents, especially in long-chain alcohols.

Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study

Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.

Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

BackgroundThe turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.ResultsThe recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.ConclusionsOur results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.

Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta.

High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZαA with the Saccharomyces cerevisiae α-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6-tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75 mg/L), a high level secretion of rFSL (310 mg/L) and tFSL (240 mg/L) was achieved providing an important improvement in enzyme production. Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4mM) and calcium ions enhanced lipase activity. During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection.

Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca

BackgroundPancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase.ResultsIn this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones.ConclusionThe fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.

N-terminal domain of turkey pancreatic lipase is active on long chain triacylglycerols and stabilized by colipase.

The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin–water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.

Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris

Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2-V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent.

Biochemical and molecular characterization of a lipase from an Algerian isolated Staphylococcus aureus strain

A Staphylococcus aureus strain, isolated from an Algerian biotope, secretes a non‐induced lipase in the culture medium. The S. aureus lipase (SAL) was purified to homogeneity. Pure SAL is a monomeric protein (43 kDa). The 20 N‐terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. SAL presents specific activities of about 1600 and 555 U mg−1 using tributyrin and olive oil emulsion as substrates, respectively. In contrast to other staphylococcal lipases previously characterized, SAL was stable at a pH range from 6 to 9 after 1 h incubation, and retained 50% of its activity after 10 min incubation at 50 °C. The purified enzyme was also characterized using monolayer technique. Lipase activity can be measured only when the surface pressure exceeds 15 mN m−1. The critical surface pressure (πc) measured with egg‐PC films was estimated at 33 mN m−1. SAL showed a preference for the distal ester groups of the diglyceride isomers at low surface pressure, for the adjacent ester groups at high surface pressure and a preference for the sn‐3 position of the 2,3‐sn‐enantiomer of dicaprin. Cloned and sequenced gene part, encoding the mature lipase shows, in comparison with S. aureus lipase 3 (SAL3), a deletion of three residues (LKA) at the N‐terminal extremity and a substitution of glycine 208 and isoleucine 226 with an arginine and leucine, respectively.

Evaluation of the recombinant turkey pancreatic lipase phospholipase activity: A monolayer study.

Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols.