An Alkalothermophilic Amylopullulanase from the Yeast Clavispora lusitaniae ABS7: Purification, Characterization and Potential Application in Laundry Detergent

In the present study, α-amylase and pullulanase from Clavispora lusitaniae ABS7 isolated from wheat seeds were studied. The gel filtration and ion-exchange chromatography revealed the presence of α-amylase and pullulanase activities in the same fraction with yields of 23.88% and 21.11%, respectively. SDS-PAGE showed a single band (75 kDa), which had both α-amylase (independent of Ca2+) and pullulanase (a calcium metalloenzyme) activities. The products of the enzymatic reaction on pullulan were glucose, maltose, and maltotriose, whereas the conversion of starch produced glucose and maltose. The α-amylase and pullulanase had pH optima at 9 and temperature optima at 75 and 80 °C, respectively. After heat treatment at 100 °C for 180 min, the pullulanase retained 42% of its initial activity, while α-amylase maintained only 38.6%. The cations Zn2+, Cu2+, Na+, and Mn2+ increased the α-amylase activity. Other cations Hg2+, Mg2+, and Ca2+ were stimulators of pullulanase. Urea and Tween 80 inhibited both enzymes, whereas EDTA only inhibited pullulanase. In addition, the amylopullulanase retained its activity in the presence of various commercial laundry detergents. The performance of the alcalothermostable enzyme of Clavispora lusitaniae ABS7 qualified it for the industrial use, particularly in detergents, since it had demonstrated an excellent stability and compatibility with the commercial laundry detergents.

Studies on the Thermal Stability of Peroxidase from Leaf of Oil Palm (Elaeis guineensis)

Peroxidase was extracted from leaves of oil palm tree with 0.01M phosphate buffer pH 7.0. It was partially purified using 70% ammonium sulphate ((NH4)2SO4) precipitation. This resulted in peroxidase with activity of (26U/ml) and specific activity of 35.8U/mg. Effect of heat on the activity of peroxidase was studied at temperature of 323-363°K. After gel filtration on sephadex G100, the peroxidase activity increased to 27U/ml, with specific activity of 55U/mg .The overall purification fold was 4 with 51.9% enzyme recovery. The peroxidase partially purified from leaves of oil palm tree showed pH and temperature optima of 5.0 and 50°C respectively. High pH and temperature stabilities of pH 5.0 to pH 9.0 and 50°C to 70°C were obtained respectively. Also, the activation energy (Ea) of the reaction was - 21.616kj/mol. The free energy changes (ΔG) were 96008.64, 96315.59, 97901.63, 94132.33 and 97146.75kj/mol at 323,333,343,353 and 363°K respectively. It was observed that the D-values were decreasing with increasing temperature with a Z-value of 0.044. The enthalpy results suggest that the reaction was exothermic, non-spontaneous and reversible.

Acid Stable Yeast Cell-Associated Tannase with High Capability in Gallated Catechin Biotransformation

Previously, nine tannin-tolerant and tannase-producing yeasts were isolated from Miang; all produced cell-associated tannase (CAT) during growth in tannin substrate. Among which, only CAT from Sporidiobolus ruineniae showed better stability than its purified form. Yet, it is of particular interest to directly characterize CATs from the latter yeasts. In this study, four CATs from yeasts, namely Cyberlindnera rhodanensis A22.3, Candida sp. A39.3, Debaryomyces hansenii A45.1, and Cy. rhodanensis A45.3 were characterized. The results indicate that all CATs were produced within the same production yield (11 mU/mL). Most CATs exhibited similar pH and temperature optima and stabilities, except for CAT from Cy. rhodanensis A22.3. This CAT was assigned as acid-stable tannase due to its unusual optimum pH of 2.0 with pH stability and half-life thermostability in the range of pH 2.0–4.0, and 70 °C, respectively. All CATs demonstrated high substrate specificity toward epigallocatechin gallate and epicatechin gallate, thus forming epigallocatechin and epicatechin, respectively. Moreover, they showed operational stability to repeated use for up to five cycles without loss of the initial activity. Therefore, CATs from these yeasts could be useful for the extraction and biotransformation of tea catechins and related applications.

Two marine GH29 α-L-fucosidases from an uncultured Paraglaciecola sp. specifically hydrolyze fucosyl-N-acetylglucosamine regioisomers

L-Fucose is the most widely distributed L-hexose in marine and terrestrial environments, and presents a variety of functional roles. L-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae, and is found in human milk oligosaccharides and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-L-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in E. coli. All five enzymes (Fp231, Fp239, Fp240, Fp251, Fp284) hydrolyzed the synthetic substrate CNP-α-L-fucose. By screening each of these enzymes against up to 17 fucose-containing oligosaccharides Fp231 and Fp284 showed strict substrate specificities against the fucosyl-N-acetylglucosamine regioisomers Fuc(α1,4)GlcNAc and Fuc(α1,6)GlcNAc, respectively, the former representing a new specificity. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s−1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

Non-Enzymatic Glycation and Formation of Advanced Glycation End-Products Alters the Activity and Related Kinetic Properties of Aldose Reductase

Aldose reductase was incubated with and without either fructose or glucose for 42 days to initiate the glycation process. The concentrations of fructosamine were measured on every 7th day using the standard nitroblue tetrazolium reagent assay. Carboxymethyllysine formed was determined using enzyme linked immunosorbent assay-based methods, fluorescent end-products were measured using spectrofluorometric methods. Activities were assayed by measuring the absorbance of co-factor nicotinamide adenine dinucleotide phosphate hydrogen at 340 nm. Fructosamine, carboxymethyllysine protein adducts and fluorescent end-products were significantly higher (p < 0.001) when aldose reductase was incubated with fructose or glucose than without. Although the glycation of aldose reductase did not result in the alteration of both the optimum pH and temperature of the enzyme, both the activity and Vmax were increased, whereas Km was decreased. Non-enzymatic glycation of aldose reductase increases both its activity and Vmax, while decreasing its Km. Additionally, glycation did not affect the pH of enzyme and temperature optima.

Thermal performance of marine diatoms under contrasting nitrate availability

Environmental factors that interact with increasing temperature under the ongoing global warming are an urgent issue determining marine phytoplankton’s performance. Previous studies showed that nutrient limitation alters phytoplankton responses to temperature and may lower their temperature optima (Topt), making them more susceptible to high temperatures. The generality of this relationship is unknown, as very few species were tested. Here we investigated how growth rate depended on temperature at two contrasting nitrogen concentrations in six marine diatoms isolated from different thermal environments, including the tropics. Low nitrate had a significant effect on thermal performance in five of the six species. The effect size was larger around the optimum temperature for growth, resulting in flattened thermal performance curves but no shift in Topt. While that trend is independent of the thermal regime from which each species was isolated, the implications for the phytoplankton response to global warming may be region dependent.