Purification and Characterization of Superoxide Dismutase Enzyme from Punica granatum L.

Superoxide dismutase (SOD) constitutes a very important antioxidant defense against oxidative stress in the body. SOD is found aplenty in many organisms, from microorganisms to plants and animals. Pomegranate (Punica granatum L.) peels and fruits have been used in traditional medicine. In this study, SOD (E.C 1.15.1.1) was purified from Punica granatum L. For this purpose, Punica granatum L. was homogenized centrifuged, fractioned with ammonium sulfate to precipitate, and then DEAE chromatography separation was applied. Punica granatum L. was purified 16.60-fold. SOD enzyme-specific activity was found as 166 U/mg protein.

The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain

Aim: The confirmation of lipolytic activity and role of Rv1900c in the mycobacterium physiology Methods: rv1900c/N-terminus domain ( rv1900NT) were cloned in pET28a/ Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressing rv1900c/ rv1900NT altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in intracellular survival of bacteria.

Beware, commercial chondroitinases vary in activity and substrate specificity

Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play key roles in health and disease. The most common method to analyze their structure is by digestion into disaccharides with bacterial chondroitinases, followed by chromatography and/or mass spectrometry. While studying the structure of oncofetal CS, we noted a large variation in the activity and specificity of commercially available chondroitinases. Here studied the kinetics of the enzymes and used high-performance liquid chromatography–mass spectrometry to determine the di- and oligosaccharide products resulting from the digestion of commercially available bovine CS A, shark CS C and porcine DS, focusing on chondroitinases ABC, AC and B from different vendors. Application of a standardized assay setup demonstrated large variations in the enzyme-specific activity compared to the values provided by vendors, large variation in enzyme specific activity of similar enzymes from different vendors and differences in the extent of cleavage of the substrates and the generated products. The high variability of different chondroitinases highlights the importance of testing enzyme activity and monitoring product formation in assessing the content and composition of chondroitin and DSs in cells and tissues.

Antioxidant Capacity and Behavioral Relevance of a Polyphenolic Extract of Chrysanthellum americanum in a Rat Model of Irritable Bowel Syndrome

Chrysanthellum americanum L. (Vatke) is a medicinal plant from the Compositae family used in west-African traditional medicine, known for its flavonoid and saponin richness and for its strong antioxidant potential. In the present study, we assessed the effects of Chrysanthellum americanum polyphenolic extract in the psychological stress-induced rat model of irritable bowel syndrome (IBS), a chronic functional digestive tract disorder marked by immune and inflammatory-related disturbances of central nervous and peripheral intestinal systems, which is often associated with mood disorders including depression and anxiety. Consequently, memory impairment, anxiety and depression behavioral indicators, and cerebral oxidative stress biomarker dynamics were evaluated in a multifactorial heterotypic stress-exposed IBS rats after 6-day gavage with polyphenolic C. americanum extract (100 mg/kg body weight). Y-maze, elevated plus maze, and forced swimming tests were used for assessing behavioral responses. Administration of the extract exhibited significant anxiolytic and antidepressant-like effects coupled with significantly increased temporal lobe antioxidant enzyme specific activity (superoxide dismutase and glutathione peroxidase) and decreased malondialdehyde levels, a well-known lipid peroxidation marker. Furthermore, linear regression statistical analyses showed significant correlations between the oxidative stress parameters and behavioral tests. In conclusion, our results suggest that the administration of Chrysanthellum americanum polyphenolic extract could ameliorate mood and cognitive disturbances related to stress-induced in an IBS rat model. This could be also related to cerebral oxidative stress status attenuation.

Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability

Alginate lyase degrades alginate by the β-elimination mechanism to produce oligosaccharides with special bioactivities. The low thermal stability of alginate lyase limits its industrial application. In this study, introducing the disulfide bonds while using the rational design methodology enhanced the thermal stability of alginate lyase cAlyM from Microbulbifer sp. Q7. Enzyme catalytic sites, secondary structure, spatial configuration, and molecular dynamic simulation were comprehensively analyzed. When compared with cAlyM, the mutants D102C-A300C and G103C-T113C showed an increase by 2.25 and 1.16 h, respectively, in half-life time at 45 °C, in addition to increases by 1.7 °C and 0.4 °C in the melting temperature, respectively. The enzyme-specific activity and kcat/Km values of D102C-A300C were 1.8- and 1.5-times higher than those of cAlyM, respectively. The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase.

Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the xylooligosaccharide types from xylan hydrolysis by this enzyme. Based on this research, the optimum time for enzyme production occurred at 96 hours with the enzyme activity of 6.275 U/mL and enzyme specific activity of 5.093 U/mg. The specific activities were obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0 U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram showed that the molecular weight of xylanase protein were about 25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji hidrolisis untuk mengetahui jenis xilooligosakarida yang dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96 dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil pemekatan dengan amicon® ultra-15 centrifugal filter devices, kromatografi filtrasi gel dan kromatografi penukar anion mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

PEMANFAATAN PERLIT YANG TELAH DIMODIFIKASI UNTUK AMOBILISASI ENZIM BROMELAIN

ABSTRACT Amobilization of bromelaine enzyme extracted from ananas fruit (Ananas comusus) acetone has been done. Modified perlite in the amino silica phosphat (ASP) is used as matrix amobilization. The protein content has determined by Lowry method, while enzyme activity were determined by Anson method caseine as substrate. It native enzyme specific activity was 0.1281 unit/mg with konsentration 20000 ppm, pH 7.0, incubation time 35 minutes, and temperature 40°C. Amobil bromelaine specific activity was 0.7438 units/mg with substrate on 20000 ppm pH 7.0, incubation time 30 minutes and temperature 37°C. This bromelaine enzymes activity was increased six times than native enzim and showed the activity for several repeatation. Keywords: perlit, enzim bromelain, amobilization

PRODUKSI DAN PENENTUAN KONDISI OPTIMUM ENZIM XILANASE Bacillus amyloliquefaciens FUKUMOTO PADA SUBSTRAT XILAN JERAMI

ABSTRACT The production and determination of optimal condition of xylanase produced by Bacillus amyloliquefaciens on rice straw xylan were investigated in this study. The parameters to be observed were optimal conditions of pH, temperature, substrate concentration and incubation time. Xilanase activity was determined by measuring the amount of reducing sugar formed in the enzymatic reaction based on Somogyi Nelson method. Optimal conditions needed for the production of xylanase were at pH 7, temperature 27°C and six days of incubation time. While optimal conditions of xylanase action were reached at pH 8.2, temperature 45°C, substrate concentration 3.5%(w/w) and 15 minutes of incubation time with enzyme activity and enzyme specific activity of 1.285 U/mL and 0.738 U/mg respectively. As a comparison, xylanase was also produced on pure xylan (birchwood), enzyme activity and enzyme specific activity obtained were 2.701 U/mL and 1.658 U/mg respectively. Cellulase content in enzyme produced on rice straw xilan showed the enzyme activity of 0.094 U/mL. Keywords : xylanase, Bacillus amyloliquefaciens, rice straw xilan

Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion ofArabidopsisCellulose Synthase-Like A2 Inserted into Golgi Membrane

Cellulose synthase-like(Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily ofCslAis putatively involved in the biosynthesis ofβ-mannans. Here we report a study on the cellular localization and the enzyme activity of anArabidopsisCslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out thein vitrosynthesis of a14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.