Molecular Weight Of Enzyme Research Articles

PRODUCTION, PURIFICATION AND DETERMINATION MOLECULAR WEIGHT OF COAGULASE ENZYME FROM STAPHYLOCOCCUS AUREUS ISOLATED FROM CLINICAL SAMPLES

Background: Coagulase is a critical enzyme produced by pathogenic strains of Staphylococcus aureus, which plays a significant role in the classification of staphylococci into coagulase-positive (CoP) and coagulase-negative staphylococci (CoNS). This enzyme interacts with prothrombin to induce blood clotting, which is essential for understanding its pathogenic mechanisms. Knowledge Gap: Despite its importance, detailed methods for extracting, purifying, and characterizing coagulase from clinical S. aureus isolates remain underexplored, particularly in relation to optimizing purification conditions and accurately determining its molecular weight. Aims: This study aimed to extract and purify coagulase from Staphylococcus aureus isolated from clinical samples and to determine the enzyme's molecular weight using SDS-PAGE. Methods: A total of 2,000 clinical samples were collected from hospitals in Baghdad, yielding 130 isolates of S. aureus. The optimum conditions for coagulase production were identified as pH 7.5 and 37°C. Coagulase was extracted and purified through ammonium sulfate precipitation (50-80% saturation), SDS-PAGE, ion exchange chromatography with DEAE cellulose, and gel filtration using Sephadex G150. Results: The crude coagulase extract exhibited an activity of 1.7 U/ml. Following purification, the enzyme's specific activity was measured, and the molecular weight of the coagulase was determined to be 36 kilodaltons (kDa). Novelty and Implications: This study provides a detailed protocol for the extraction and purification of coagulase from clinical isolates of S. aureus, along with the molecular weight determination of the enzyme. The findings enhance the understanding of coagulase's biochemical properties and its role in staphylococcal pathogenicity, potentially contributing to improved diagnostic and therapeutic strategies.

Characterization of raw starch hydrolysing and detergent-compatible amylase from newly isolated Bacillus subtilis from bakery chimney

Amylases are highly needful product in food, beverage, and detergent industry. With the objective of isolating α-amylases producing strains, samples were collected from local bakery chimneys and kitchens, Chennai, Tamil Nadu, India. The potential α-amylase producers were isolated and screened by Starch-Iodine plate assay method. The optimized parameters such as 1.5% of starch concentration in the media with 7.2-pH at 35 °C for 36 h enhanced the production of α-amylase. The strain showing high potential, identified by 16s rRNA sequencing was found to be Bacillus subtilis. The Bacillus subtilis strain produced extracellular α-amylase with activity of 1330.14 U/mL. Further partial purification by ammonium sulphate precipitation followed by dialysis and column chromatography enhanced the specific activity to 1695.306 U/mg. The molecular weight of enzymes was found to be around 36 kDa. The enzyme stability and activity characterization revealed that the enzyme was thermotolerant which was able to retain more than 70% activity at 70 °C, 80 °C and 90 °C. The purified enzyme was also found to be Ca2+ independent. The hydrolysis of raw starch from rice, wheat and corn flour using the purified α-amylase from Bacillus subtilis showed significant activity of 72.8%, 76.1% and 65.2% with 4 h incubation respectively. The potentiality of the enzyme as an additive with commercial detergent was also determined to be effective. This study favourably attracts the attention towards industrial applications.

Secretion of the cytoplasmic and high molecular weight β-galactosidase of Paenibacillus wynnii with Bacillus subtilis

BackgroundThe gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa.ResultsIn this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%.ConclusionFor the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

ВЫДЕЛЕНИЕ ДЕГАЛОГЕНАЗ ПЕРФТОРКАРБОНОВЫХ КИСЛОТ ШТАММОВ PSEUDOMONAS PLECOGLOSSICIDA 2,4 Д И PSEUDOMONAS MOSSELII 5(3)

The aim of this study was to isolate and investigate enzymes involved in the biodegradation of perfluorocarboxylic acids (PFCAs) in Pseudomonas plecoglossicida strain 2,4-D and Pseudomonas mosselii strain 5(3). In this research, enzymes from these strains were concentrated from the culture medium by ultrafiltration and ammonium sulfate precipitation, followed by purification through size-exclusion chromatography. The enzyme activity after ultrafiltration was higher Enzymes from strain 2,4-D released 2 ppm of fluoride ions after 24 hours, increasing to 40,7 ppm after 48 hours. For strain 5(3), fluoride release reached 3 ppm at 24 hours and 46 ppm at 48 hours, confirming the activity of the isolated dehalogenases and their ability to degrade PFCAs. Size-exclusion chromatography revealed that the molecular weight of enzymes from strain 5(3) is approximately 80 kDa, while for strain 2,4-D, it is 76 kDa. The study of these enzymes holds both applied significance for environmental remediation technology development and fundamental importance. Understanding the molecular structure and mechanisms of dehalogenase activity not only provides insights into the cleavage of robust C-F bonds but also sheds light on enzyme evolution as a whole.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50°C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20°C for 12weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Biofuel Production from Mango and Orange Peel and Tapioca Shells by Fermentation Using Consortium of Bacteria: Agricultural and Food Waste Valorization

Lignocellulosic substrates are considered to be crucial substrates for the production of biofuels. The main objective of the study is to attempt to produce bioethanol using bio-wastes such as mango peels, orange peels, and tapioca shells as renewable sources by employing three bacteria viz., Enterobacter cloacae (ICBP1), Pseudomonas aeruginosa (ICBP7), and Bacillus cereus (ICBP15), which were chosen to produce cellulase enzymes using the submerged fermentation method, which is a novel method for the production of bioethanol. The “zone of clearance” in bacterial growth on CMC agar plates determined the choice. The mixed culture infected units produced a more reduced sugar, i.e., the presence of aldehyde and ketones except sucrose. At 72 h, greater than 41.0 ± 0.48 mL and 0.83 ± 0.07% of ethanol was recovered. This contrasts with the reduced quantities at 24 and 48 h. SDS-PAGE examination showed that the three cellulose-producing bacterial strains (ICPB1, ICPB7, and ICPB15) had enzyme molecular weights of 80–100, 20–30, and 14–20 kDa, respectively, compared to the other 17 isolates. Fourier-transform infrared (FTIR) spectroscopy was used to estimate the bioethanol. The spectrum bands from 1700 to 1800 cm−1 showed bioethanol’s unique absorption characteristics, and GC-MS confirmed 31.38% ethanol. The findings of the research demonstrate that the utilization of fermentation technology, specifically employing microbes, to produce bioethanol from bio-wastes such as fruits and vegetables has the potential to address the worldwide fuel energy requirements.

Regulation and Characterization of Amylase Enzyme Secretion In the Digestive Tract of <i>Antheraea Assamensis</i> Helfer

Antheraea assamensis Helfer is an endemic non-mulberry lepidopteran species of North East India with high commercial demand for its golden hued silk. Antheraea assamensis suffers from a protozoan disease pebrine which lead to death of the silkworm. In this study, the effect of various factors (starvation, feeding, temperature, pH, Ca ion and larval stage) in the regulation of amylase secretion is compared in healthy and diseased muga silkworm. Moreover, enzyme zymography and purification of amylase from the midgut is also performed. Result shows that, the secretion of amylase in Antheraea assamensis in starved condition, significantly decreases. Refeeding experiments (after 2 days of starvation) suggest that amylase secretion sharply increases due to feeding stimulus. The purified enzyme molecular weight is confirmed as 56 kDa by SDS PAGE. The purification fold of purified enzyme is 34 times higher than the crude enzyme extract.

Ginsenoside F1 preparation from ginsenosides Re and Rg1 mixture of ginseng leaves by a 6-O-glycoside-ginsenosidase from Aspergillus sp. g383 strain

Ginsenoside F1 was prepared from ginseng-leaf ginsenosides Re and Rg1 using a 6-O-glycoside-ginsenosidase from Aspergillus sp.g383 strain. From 500 g ginseng leaves, a 25.1 g mixture of 54.2% Re and 45.8% Rg1 were extracted. The enzyme molecular weight was 54.5 kDa; the optimal temperature was 45 °C; and optimal pH, 5.0; Re's V max and Km were 11.6 mM/h and 10.9 mM, respectively; Rg1's were 7.69 mM and 0.917 mM/h. The 6-O-glycoside-ginsenosidase conversion rates of Re and Rg1 into F1 were faster than those of the other enzymes. In the preparation of ginsenoside F1, 2.5% mixture of Re and Rg1 from ginseng leaf-part was reacted at 45 °C for 36 h by crude enzyme at a low cost. From a 25 g mixture of Re and Rg1, 13.8 g ginsenoside F1 was obtained. The purity of F1 was 95%, and the molar yield from Re and Rg1 was 75%. F1 weight-yield from ginseng-leaves was 2.76%. Furthermore, ginseng-leaves were used to obtain extra ginsrnoside Re and ginseng-leaf PPD-ginsenosides. Thus, the ginsenosides F1, which could be used safely in ginseng food, cosmetics or drugs, were successfully produced by a non-GMO crude-enzyme from a Re and Rg1 mixture of ginseng-leaves.

Efficient three phase partitioning of actinidin from kiwifruit (Actinidia deliciosa) and its characterization

Three phase partitioning (TPP) method was effectively utilized for the extraction and purification of milk clotting protease (actinidin) from the kiwifruit pulp. The different purification parameters of TPP such as ammonium sulfate saturation, ratio of the crude kiwifruit extract to tert-butanol, and the pH value of extract were optimized. The 40% (w/v) salt saturation having 1.0:0.75 (v/v) ratio of crude kiwifruit extract to tert-butanol at 6.0 pH value exhibited 3.14 purification fold along with 142.27% recovery, and the protease was concentrated exclusively at intermediate phase (IP). This fraction showed milk-clotting activity (MCA), but there was no such activity in lower aqueous phase (AP). The enzyme molecular weight was found to be 24 kDa from Tricine SDS-PAGE analysis. Recovered protease demonstrated greater stability at pH 7.0 and temperature 50 °C. The V max and K m values were 121.9 U/ml and 3.2 mg/ml respectively. Its cysteine nature was demonstrated by inhibition studies. This study highlighted that the TPP is an economic and effective method for extraction and purification of actinidin from kiwifruit, and it could be used as a vegetable coagulant for cheesemaking.

Purification and anticancer activity of glutaminase and urease free intracellular l-asparaginase from Chaetomium sp.

l-asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. l-asparaginase helps in removing acrylamide found in fried and baked foods which is carcinogenic in nature. The search for new therapeutic enzymes is of great interest in both medical and food applications. The present work aims to isolate the intracellular l-asparaginase from endophytic fungi Chaetomium sp. The intracellular enzyme was partially purified by chromatographic techniques. Molecular weight of enzyme was found to be ~66 kDa by SDS PAGE analysis. The enzyme is highly specific for l-asparagine and did not show glutaminase and urease activity. Maximum enzyme activity was found to be 58 ± 5 U/mL at 40 °C, pH 7.0 with 2 μg of protein. Intracellular l-asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells.

Degradation mechanism and toxicity reduction of methyl orange dye by a newly isolated bacterium Pseudomonas aeruginosa MZ520730

Methyl orange (MO) dye is recalcitrant in nature, hard to degrade and if released into the soil and aquatic resources could cause serious threats on environment and human health. MO is toxic to plant growth. Bacterial treatment may be a sustainable solution for its degradation and decolourization. In this work, a bacterium (RKS6) was isolated from textile industry wastewater and sludge samples and identified as Pseudomonas aeruginosa based on the 16S rRNA gene sequencing analysis. RKS6 showed more than 99% decolorization of MO dye (100 mg/l) and 96% reduction of total organic carbon (TOC) within 12 h, at 30 °C, pH 7 at static conditions. RKS6 also produced MnP enzyme of molecular weight ∼53 kDa as characterized by the SDS-PAGE analysis. Further, LC-MS analysis showed that MO dye was degraded into 4-[(4-aminophenyl) diazenyl] benzene sulfonate, 4, 2-((dihydroxymethyl) hyrazono-4) 5-benzene sulfonate, 4-(triazan-2-yl) benzene sulfonic, water and carbon dioxide by RKS6. Toxicity assessment showed that the solution treated by the bacterium allowed 90% seed germination indicating that RKS6 was effective in mineralization and detoxification of MO dye and can be effectively used in industrial wastewater treatment.

Production, Purification and Characterization of Xylanase Enzyme from <i>Bacillus sp</i> in Solid State Fermentation

Xylanases are extensively applied in paper and pulp industries as well as during preparation of baked products to improve their quality. Additionally, it is also used in coffee, oil and starch industries in order to increase their nutritional values. Soil samples were collected near saw mills in various localities of Bangalore urban to isolate organisms for the production of xylanase using solid state fermentation. Six organisms were isolated using selective media based on their morphological characters. Among them one organism showed maximum production of xylanase enzyme identified as Bacillus sp based on their biochemical test and 16s RNA sequencing. Solid substrate fermentation was carried out using various agro wastes such as sugar cane bagasse, saw dust, paddy husk, wheat straw and orange peel powder. Sugarcane bagasse showed maximum production of enzyme compared to other substrates with different physical parameters such as pH 8, temperature at 35 °C after 72 hrs of incubation. Trace element such as Mg++ enhances the production of enzyme more than 22 % compared with other metal ions like Ca++, Mn++ and Fe++. After production, enzyme purified by using three step methods such ammonium sulphate precipitation, dialysis, ion exchange and gel filtration. Fold purification was increased up to 12 fold, yield 36 % and molecular weight of enzyme was 62 KDa determined using SDS PAGE.

Efficient degradation and detoxification of methylene blue dye by a newly isolated ligninolytic enzyme producing bacterium Bacillus albus MW407057

In present work, a LiP enzyme producing bacterium was isolated form textile wastewater and sludge sample and identified as Bacillus albus by 16S rRNA gene sequencing analysis. This bacterium decolorized 99.27 % MB dye and removed 83.87 % COD within 6 h at 30 °C, pH 7, 100 rpm and 100 mg/l of dye concentration in presence of glucose and yeast extract as carbon and nitrogen source, respectively. The bacterium also produced LiP enzyme of molecular weight ∼48 kDa, characterized by SDS-PAGE analysis. Different metabolites like monomethylthionine, thionin, (E)-2-(3-Oxopropylidene)-2H-benzo[b][1,4] thiozine-3-carboxylic acid, N-(3,4-dihydroxyphenyl)-N-methylformamide, ethylamine, water and carbon dioxide produced during treatment process were characterized by FT-IR and LC–MS analysis. Further, the toxicity assessment results showed that the toxicity of bacteria treated dye solution was reduced significantly allowing 90 % seed germination indicating that the isolated bacterium B. albus has high potential to decolorize and detoxify MB dye for environmental safety.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using β-galactosidase as a model

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using β-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant β-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized β-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant β-galactosidase, showing its potential application in other high-molecular-weight enzymes.

3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

Identification of a novel tailor-made chitinase from white shrimp Fenneropenaeus merguiensis

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed KM, Vmax and kcat values of 78.18 μM, 0.07261 μM. min−1 and 43.37 s−1, respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste.

Isolation and Purification of Organophosphorus Hydrolases Secreted from Acetone-acclimated Phosphorus Accumulating Organisms and Study of Their Properties for Hydrophobic Organophosphorus Sensor.

The present work studied an acclimation method for phosphorus accumulating organisms (PAOs) with a high content of acetone in culture solutions to develop microbial-based enzyme sensors for highly hydrophobic organophosphorus (OP) pesticides. Through three steps of cultivation and acclimation, only rod-shaped bacteria survived among the various PAOs. The extracellular enzymes released from the acclimated PAOs were salted out by using ammonium sulfate, then purified by a dialysis membrane and a DEAE-Sepharose FF anion exchange column. Two enzyme components were successfully separated-both of which showed hydrolase activity on disodium p-nitrophenyl phosphate (enzyme I, 1.57 μmol/(min·μg); enzyme II, 0.88 μmol/(min·μg) at 45°C). Further, SDS-PAGE gel electrophoresis results showed that the molecular weights of enzymes I and II were about 15.11 and 11.98 kDa, respectively. On this basis, the applicability of the enzyme in hydrophobic OP biosensors was demonstrated.

Purification and characterization of novel α-amylase from Anoxybacillus ayderensis FMB1

The thermophilic amylase producing Anoxybacillus ayderensis FMB1 was isolated from Sorgun Hot Spring in Turkey. In order to obtain improved production of the enzyme for cost-effective process, optimization of the process parameters is required. Optimum conditions for amylase production were determined using the traditional Submerged Fermentation (SmF) method. The maximum amylase activity was 295.8 U/mg, under the optimized conditions (24-h, 55 °C, and pH 8.0) in liquid BMB medium. The best enzyme production was obtained in the presence of 1.5% soluble starch as carbon source and 1% yeast extract as nitrogen source. Enzyme was produced under optimal conditions and partially purified. Partially purified enzyme was stable for up to 120 minutes at 50 °C, and its stability was found to be pH 5.0–10.0. Regarding the effects of some metals and chemicals on the activity of the partially purified enzyme, Ca2+ increased the activity by 21%, but enzyme activity was decreased by Mg2+, Zn2+, Ni2+, and Cu2+ by 5%, 29%, 57%, and 64%, respectively. and the enzyme activity was completely inhibited by Hg2+. In addition; DTT, PMSF, PCMB, SDS and EDTA caused a decrease in enzyme activity by 3%, 4%, 12%, 27% and 77%, respectively. The molecular weight of enzyme was found to be 58.5 kDa by SDS-PAGE. The results obtained show that the thermophilic A. ayderensis FMB1 and their enzyme have biotechnological importance.

A thermophoresis–based biosensor for real–time detection of inorganic phosphate during enzymatic reactions

Inorganic phosphate (Pi)–sensing is a key application in many disciplines, and biosensors emerged as powerful analytic tools for use in environmental Pi monitoring, food quality control, basic research, and medical diagnosis. Current sensing techniques exploit either electrochemical or optical detection approaches for Pi quantification. Here, by combining the advantages of a biological Pi–receptor based on the bacterial phosphate binding protein with the principle of thermophoresis, i.e. the diffusional motion of particles in response to a temperature gradient, we developed a continuous, sensitive, and versatile method for detecting and quantifying free Pi in the subnanomolar to micromolar range in sample volumes ≤10 μL. By recording entropy–driven changes in the directed net diffusional flux of the Pi–sensor in a temperature gradient at defined time intervals, we validate the method for analyzing steady–state enzymatic reactions associated with Pi liberation in real–time for adenosine triphosphate (ATP) turnover by myosin, the actomyosin system and for insoluble, high molecular weight enzyme–protein assemblies in biopsy derived myofibrils. Particular features of the method are: (1) high Pi–sensitivity and selectivity, (2) uncoupling of the read–out signal from potential chemical and spectroscopic interferences, (3) minimal sample volumes and nanogram protein amounts, (4) possibility to run several experiments in parallel, and (5) straightforward data analysis. The present work establishes thermophoresis as powerful sensing method in microscale format for a wide range of applications, augmenting the current set of detection principles in biosensor technology.

A Thermostable and Alkalitolerant Arabinofuranosidase by Streptomyces lividus

Aim: The study aimed at producing and purifying thermostable and alkalitolerant microbial arabinofuranosidase using local Palm Kernel Cake (PKC) as substrate.
 Study Design: This is an experimental design in which samples were collected thrice and subjected to laboratory analyses from which quantitative data were obtained and analysed.
 Place and Duration of Study: Ibadan, Nigeria, Five months.
 Methodology: Bacterial strains were isolated from degrading PKC by serial dilution and pour plate technique on formulated Modified Basal Salt Agar Medium and incubated at 50°C for enzyme activity screening. Plates were afterwards flooded with 1% congo red solution for visualization of hydrolysis zone. Its arabinofuranosidase activity was optimized in solid state fermentation in PKC. Production temperature, pH, moisture content, inoculum size and agitation were studied for optimization test. Optimal production temperature and pH for arabinofuranosidase by isolate was 45°C and pH 9. Produced arabinofuranosidase was purified to apparent homogeneity with ammonium sulphate precipitation, dialysis and column chromatography techniques. Stability of arabinofuranofuranosidase obtained to temperature, pH, substrate concentration and some ions was determined as well as its molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
 Results: Isolate with highest arabinofuranosidase activity was selected and identified as Streptomyces lividus. Purity level attained was 16.36 fold. Enzyme had a specific activity of 25.4 U/mg, and total enzyme activity of 13.2 U. Molecular weight of enzyme appeared as a band of 30 kDa. Purified arabinofuranosidase enzyme revealed optimum temperature and pH as 60oC and 9 respectively. Enzyme was stable over a broad pH range of 3-11, and temperature of 30-80oC. Residual activity after incubating for 1 hour at 70oC was 64%. Enzyme kinetics studies showed Km and Vmax values for P-nitrophenyl arabinofuranoside were 2.3mM and 0.7U/min respectively.
 Conclusion: Apart from Solid State Fermentation (SSF) of PKC being a potential fermentation technique for production of arabinofuranosidase by Streptomyces lividus, the enzyme was highly stable.