Production and characterization of intracellular invertase from Saccharomyces cerevisiae (OL629078.1), using cassava-soybean as a cost-effective substrate.

The growing global market for industrial enzymes has led to a constant search for efficient, cost-effective methods for their production. This study reports the production of invertase using inexpensive and readily available agro-materials. Starch-digesting enzymes extracted from malted unkilned sorghum were used to hydrolyze cassava starch supplemented with 2% whole soybean. The production of intracellular invertase by Saccharomyces cerevisiae OL629078.1 in cassava-soybean and yeast sucrose broth was compared. The purification and characterization of invertase produced using the low-cost medium were also reported. The results showed that there was a 4.1-fold increase in the units of invertase produced in cassava-soybean medium (318.605 U/mg) compared to yeast sucrose broth medium (77.6 U/mg). The invertase produced was purified by chromatographic methods up to 5.53-fold with a recovery of 62.6%. Estimation of the molecular weight with gel filtration indicated a molecular weight of 118 kDa. The enzyme demonstrated its maximum activity at 50 °C and there was no decrease in its activity following a 1-h incubation at this temperature. At a pH of 5.0, the enzyme demonstrated optimal activity and it maintained over 60% of its activity in the acid range (pH 3-6). The Michalis-Menten constants Km and Vmax of intracellular invertase were 5.85 ± 1.715 mM and 6.472 ± 2.099 U/mg, respectively. These results suggest that Saccharomyces cerevisiae grown on cassava-soybean is a viable, cost-effective alternative for commercial invertase production, which can be explored for biotechnological processes.

Intracellular invertase hyperproducing strain of Saccharomyces cerevisiae isolated from Abagboro palm wine.

There is an ever-increasing demand for industrial enzyme, necessitating a constant search for its efficient producers. The isolation and characterization of invertase producer yeasts from natural palm wine is reported in this study. Yeasts were isolated from fresh palm wine obtained from Abagboro community Ile-Ife, Nigeria following standard methods. A total of six yeast strains were isolated from the palm wine. The strains were screened for their ability to produce invertase and the most efficient invertase producer was characterized and identified using phenotypic and molecular methods. Isolate C showed the highest invertase activity (34.15 µmole/ml/min), followed by isolate B (18.070 µmole/ml/min) and isolate A (14.385 µmole/ml/min). The identity of isolate C was confirmed by genotypic methods to be Saccharomyces cerevisiae (OL629078.1 accession number on NCBI database). The Saccharomyces cerevisiae strain fermented galactose, arabinose, maltose, glucose, sucrose and raffinose, grew in 50% and 60% glucose and at 25-35 °C. The newly isolated Saccharomyces cerevisiae strain is an efficient producer of invertase and can be exploited for commercial biosynthesis of the enzyme for use in biotechnological applications.

Potentials of purified tyrosinase from yam (Dioscorea spp) as a biocatalyst in the synthesis of cross-linked protein networks.

We report the usefulness of yam tyrosinase as a catalyst in the synthesis of cross-linked protein networks for biopolymers. The enzyme was purified using aqueous two-phase partitioning (ATPs) and peptide mapping on SDS-PAGE was carried out to ascertain degree of similarities of tyrosinase from the yam species. The mapping revealed distinct peptide bands of 3, 4, 4 and 2 for tyrosinase from D. praehensilis, D. alata, D. rotundata and C. esculenta respectively purified using conventional method. In contrast, continuous broad band was noticed for the ATPS-purified enzymes due to bound polyethylene glycol (PEG). Tyrosinase from D. praehensilis with overall better properties was used in the synthesis of cross-linked protein networks. The enzyme catalyzed conversion of soluble proteins from whey, moringa leaves, pumpkin leaves and cow blood into fibrous (cross-linked) protein networks for improved properties and functionalities. The purified tyrosinase from D. praehensilis was also covalently bonded to bovine serum albumin (BSA) forming tyrosinase-BSA adduct with molecular weight of 118 ± 2.0 kDa, revealing its potential as a reporter enzyme by reporting BSA. The overall result further reinforces yam tyrosinase as an enzyme of interest in various biotechnological applications.

Studies on peroxidase from Moringa oleifera Lam leaves.

Kinetic and physicochemical properties of Moringa oleifera peroxidase purified using a novel and cost efficient protocol was investigated with a view to providing information on its possible biotechnological potentials. Moringa oleifera peroxidase was purified to homogeneity in two steps, involving ATPS and size exclusion chromatography on Sephadex G-100 with a yield of 84.12 %. In-gel activity staining revealed the presence of one isoform of peroxidase. The purified peroxidase is monomeric with native and subunits molecular weight of 38.9 and 43.5 kDa respectively. Kinetic parameters - V max, K m(app) o-dianisidine, K m(app) H 2 O 2 of the purified enzyme were 2.5 units/mg protein, 0.020 ± 0.04 mM and 1.37 ± 0.18 mM respectively. Its optimum pH and temperature were 5 and 30 °C respectively. The purified enzyme cross-linked BSA into an insoluble matrix with the aid of caffeic acid. The study concluded that the purification scheme adopted is rapid and efficient, the purified enzyme exhibited some physiochemical properties that make it suitable for various biotechnological applications.

Induction and catalytic properties of grasshopper (Zonocerus variegatus) glutathione transferase fed on different food plants.

In order to establish the role of diet on the induction and catalytic properties of glutathione transferase (GST) in insects, variegated grasshopper (Zonocerus variegatus) was exposed to different food plants separately for 30 days and the properties of the induced enzyme were then investigated. Insects fed on cassava (M. esculenta) leaves had the highest GST induction followed by insects fed on bitter leaf (V. amygdalina). Z. variegatus that fed in the wild on different food plants had the least suggesting that allelochemicals in the food plants have a compensatory toxicity-alleviating actions on one another. 1-Chloro-2,4-dinitrobenzene (CDNB) was the best substrate for all the induced GST however, the mode of binding of the substrate to the induced enzyme was not the same. GST from M. esculenta-fed insect showed ping-pong kinetic mechanism whereas GSTs from V. amygdalina and T. procumbens-fed insects showed random sequential mode of substrate binding. Catalytic efficiency (kcat/Km) of GST from M. esculenta-fed insects was 3-8-fold higher than other induced enzymes. Commercial insecticides- cypermethrin and lindane had an inhibition constant, Ki, of 0.13±0.004 mM and 0.68±0.09 mM, respectively, suggesting that the concentration as used in the field (0.03 mM for cypermethrin and 0.3 mM for lindane) would have little effect on the insect's GST. The study concluded that higher GST activity are induced in insects that fed on monotonous diets than those that fed on various food plants. Hindgut appears to be the primary organ of detoxication. The catalytic properties of the induced enzymes are different from one another.

Physicochemical properties of free and immobilized tyrosinase from different species of yam (Dioscorea spp).

A shortened method of purification and immobilization of tyrosinase from different species of yam (Dioscorea spp) on insoluble supports is described. The enzyme was purified by aqueous two-phase partitioning (ATPS) followed by gel filtration chromatography. The purified enzyme was immobilized on Ca-alginate, polyacrylamide gel or as cross-linked enzyme aggregate (CLEA) to obtain a yield of between 51-64%, 33-46% and 52-65% respectively for all the yam species. The optimum pH obtained for tyrosinase immobilized on polyacrylamide gel and CLEA was equivalent to that of free enzyme (pH 6.5). In contrast, Ca-alginate entrapped tyrosinase exhibited a shift of optimum pH to 7.0. Entrapped Tyrosinase in polyacrylamide gel and Ca-alginate also retained the same optimum temperature as the free enzyme (50 °C). While the optimum temperature of CLEA shifted to 60 °C. When subjected to four repeated use cycles, tyrosinase entrapped in polyacrylamide gel, Ca-alginate and CLEA still retained close to 40, 35 and 45 % of their initial activities respectively after the fourth cycle. The overall result further suggests yam tyrosinase as a promising enzyme for biocatalysis and biotechnological applications.

Catalytic studies of glutathione transferase from Clarias gariepinus (Burchell) in dilute and crowded solutions.

Kinetic properties of purified Clarias gariepinus glutathione transferase (CgGST) was studied in the presence of Ficoll 70, Polyethylene glycol (PEG) 6000, bovine serum albumin (BSA) and in dilute solution. This was done to mimic the cytosol thereby unraveling the actual mechanism of detoxication involving glutathione transferase (GST) in the crowded intracellular milieu. CgGST from the liver of Clarias gariepinus was purified to homogeneity by affinity chromatography on glutathione (GSH) - agarose. Initial-velocity study was performed by varying the concentrations of GSH at various fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) and vice-versa. Data obtained were fitted to the three equations representing random-ordered, compulsory-ordered and ping-pong mechanisms to obtain kinetic parameters. Product inhibition studies using sodium chloride (NaCl) was done by varying the concentrations of NaCl and CDNB at a fixed concentration of GSH and vice-versa. Data obtained were fitted to three equations representing competitive, non-competitive and uncompetitive inhibitions to obtain the inhibition constants (KiGSH and KiCDNB). Optimal temperature of CgGST activity was 20 °C both in dilute and crowded solutions. Maximum velocity (Vmax) in dilute solution was decreased, while KmGSH and KmCDNB were increased in the presence of the crowding agents. Turnover number (kcat), catalytic efficiency - kcat/KmGSH,kcat/KmCDNB and inhibition constants - (KiGSH and KiCDNB) were reduced in crowded solutions. Mechanism of catalysis was steady - state random sequential in both dilute and crowded solutions. The study concluded that although the catalytic efficiency of the enzyme was reduced in crowded solution, mechanism of catalysis remains the same in both crowded and dilute solutions.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

Preliminary studies on the renaturation of denatured catfish (Clarias gariepinus) glutathione transferase.

Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230 nm. Gdn-HCl at 0.22 M caused 50 % inactivation of the enzyme and at 1.1 M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30 µg/ml yielded 40 % percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30 % obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.

Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus.

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.

Organ distribution and kinetics of Glutathione transferase from African river prawn, Macrobrachium vollenhovenii (Herklots).

The distribution of glutathione transferase (GST) in the major organs of African river prawn (Macrobrachium vollenhovenii) was studied. All the organs studied had GST activity. The specific activity of the extract from the hepatopancreas was highest while that from the muscle lowest. Purified GST from the hepatopancreas which could conjugate glutathione (GSH) with only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl) among some electrophilic substrates tested, had a K(m)(NBDCl) of 2.2+/-0.12 mmol l(-1) while the K(m)CDNB was 2.03+/-0.29 mmol l(-1). Chloride ion, a product of the enzymatic reaction readily inhibited the conjugation of CDNB with GSH with an I50 of 0.12 mmol l(-1), whereas chloride ion up to 0.6 mol l(-1) had no inhibitory effect on the conjugation of GSH with NBDCl. However, nitrite inhibited the two reactions but the K(i) for the conjugation of NBDCl was lower than the K(i) for the conjugation of CDNB. The enzyme had an optimum temperature of 40 degrees C and an activation energy of 35.1 kJ/mol. The overall results show that M. vollenhovenii GST (mvGST) uses different mechanisms for different electrophilic substrates. The high K(m) of mvGST for the electrophilic substrates may be a special physiological adaptation for effective xenobiotic detoxication.

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfish macrobrachium vollenhovenii (herklots).

Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 +/- 1.4 kDa and a subunit Mr of 24.1 +/- 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I50 value for cibacron blue, S-hexylglutathione, hematin, and N-ethylmaleimide (NEM) were 0.01 microM, 340 microM, 5 microM and 33 mM respectively. Out of the several substrates tested, only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial-velocity studies of the enzyme are consistent with a steady-state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn-HCl) revealed that though low Gdn-HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5-4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three-state unfolding process.