Purification and some properties of chitinase fromAspergillus carneus

An extracellular chitinase fromAspergillus cerneus was purified by ammonium sulphate precipitation, gel filtration through Sephadex G-100, preparative HPLC chromatography and large slabs of polyacry-lamide gel electrophoresis.The mol wt of the enzyme was estimated to be 25000 by SDS gel electrophoresis, and it contained 9.37% (w/w) carbohydrate residue, as glucose. The pattern of its amino acid composition showed high contents of asparagine, serine, and threonine. The enzyme was active at pH 5.2 and 50°C. The K_m value of the enzyme was 4.37 mM (expressed asN-acetylglucosamine). The enzyme was stable at pH 3–9, whereas it was unstable at 70°C or more. Calcium and Mg ions slightly activated the enzyme, whereas Hg²⁺, I₂, andp-chloromercuribenzoate inhibited the enzyme activity. The enzyme hydrolyzed chitin, colloidal chitin, glycol chitin, and chitooligsac-charides, but did not hydrolyze chitosan, starch, xylan, inulin, and cellulose. The lysis ofA. niger and Micorcoccus lysodeikticus cell walls by the action of the enzyme was also investigated.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

来源于类芽孢杆菌属碱性甲壳素酶的分离纯化及其性质

甲壳素,又名几丁质(chitin),是自然界中含量仅次于纤维素的第二大天然多糖,有第六生命要素之美称.其主要存在于甲壳类动物的外壳、真菌细胞的细胞壁以及一些昆虫的外壳中,每年自然界中约有100多亿吨甲壳素生成.甲壳素是由2-乙酰氨基-2-脱氧-D-吡喃葡萄糖和2-氨基-2-脱氧-D-吡喃葡萄糖通过β-1,4糖苷键连接而成的二元线性聚合物,分子链中分布许多羟基、氨基及乙酰氨基,形成大量分子间及分子内氢键,致使其结晶度较高,化学性质十分稳定,直接利用较为困难.甲壳素不溶于稀酸、稀碱以及一般有机溶剂,工业上常用强酸强碱法处理甲壳素,以制备壳寡糖类产品,但该方法具有产品结构不单一,环境污染较为严重等缺点.甲壳素酶可特异性水解甲壳素链中β-1,4糖苷键,得到甲壳寡糖和N-乙酰氨基葡萄糖.酶解法降解甲壳素工艺简单、反应条件温和、环境友好,有很好的应用前景.我们以Paenibacillus pasadenensis CS0611为出发菌株,以蟹壳粉末为培养基唯一碳源及氮源,在适宜条件下培养48 h.发酵液经离心、硫酸铵(80％饱和度)盐析、透析除盐后得到粗酶液.再利用HiTrap DEAE FF离子交换层析和HiLoad 26/600Superdex 200 pg凝胶过滤层析对该粗酶液进行分离纯化,以得到电泳纯甲壳素酶.所制备甲壳素酶比活力为10.28 U/mg,最终纯化倍数为5.3,酶活得率为15.7％.SDS-PAGE结果表明,该甲壳素酶相对分子质量约为69 kDa.后经MALDI-TOF-MS鉴定,该酶部分肽段和来源于另一株Paenibacillus pasadenenss的甲壳素酶(accession No:gi655151624)具有较高的同源性,进一步证实所纯化蛋白为甲壳素酶.对上述纯化的甲壳素酶的酶学性质进行研究,结果发现:其最适反应温度为50℃,在20-35℃内有较好的稳定性,50℃及以上热稳定性较差;最适pH为5.0,在pH4.0-11.0间具有较高稳定性,表明该酶具有很好的耐碱性;金属离子对该酶催化活性没有明显的激活作用,表明该甲壳素酶是非金属酶.同时,对该酶的底物特异性进行研究,发现该酶对胶体甲壳素和甲壳素水解能力较强,对淀粉和纤维素无水解能力,对不同脱乙酰度的壳聚糖的水解程度随脱乙酰度不同而变化,表明该酶只能特异性识别并降解GlcNAc-GlcNAc之间的糖苷键;以胶体甲壳素为底物时,米氏常数Km为4.41 mg/mL,最大反应初速度为1.08 mg/min.利用薄板层析和高效液相色谱对酶解产物进行分析,结果表明该甲壳素酶对胶体甲壳素的降解产物主要是(GlcNAc)2.综上所述,本研究所涉甲壳素酶在甲壳二糖的酶法制备方面具有较好的应用前景.     

Purification and Characterization of a Chymosin from Rhizopus microsporus var. rhizopodiformis

Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0–8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.     

Production, Purification, and Characterization of Exoglucanase by Aspergillus fumigatus

Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K _m and V _max were 4.34 mM and 7.29 μM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.     

Bacteriocin PJ4 Active Against Enteric Pathogen Produced by Lactobacillus helveticus PJ4 Isolated from Gut Microflora of Wistar Rat (Rattus norvegicus): Partial Purification and Characterization of Bacteriocin

The increase of multidrug-resistant pathogens and the restriction on the use antibiotics due to its side effects have drawn attention to the search for possible alternatives. Bacteriocins are small antimicrobial peptides produced by numerous bacteria. Much interest has been focused on bacteriocins because they exhibit inhibitory activity against pathogens. Lactic acid bacteria possess the ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this study, an antibacterial substance (bacteriocin PJ4) produced by Lactobacillus helveticus PJ4, isolated from rat gut microflora, was identified as bacteriocin. It was effective against wide assay of both Gram-positive and Gram-negative bacteria involved in various diseases, including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus. The antimicrobial peptide was relatively heat-resistant and also active over a wide pH range of 2–10. It has been partially purified to homogeneity using ammonium sulfate precipitation and size exclusion chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of bacteriocin PJ4 purified through size exclusion chromatography resolved ~6.5 kDa protein with bacteriocin activity. The peptide is inactivated by proteolytic enzymes, trypsin, and lipase but not when treated with catalase, α-amylase, and pepsin. It showed a bactericidal mode of action against the indicator strains E. coli MTCC443, Lactobacillus casei MTCC1423, and E. faecalis DT48. Such characteristics indicate that this bacteriocin may be a potential candidate for alternative agents to control important pathogens.     

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.     

Purification and characterization of an alkaline protease prot 1 frombotrytis cinerea

Alkaline thiol protease named Prot 1 was isolated from a culture filtrate ofBotrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0–10.0 and 60°C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50°C over 120 min; it maintained 50% of activity after 60 min of heating at 60°C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25°C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.     

Collagenase produced from Aspergillus sp. (UCP 1276) using chicken feather industrial residue

An extracellular collagenolytic serine protease was purified from Aspergillus sp., isolated from the Caatinga biome in northeast Brazil by a two‐step chromatographic procedure, using an anion‐exchanger and gel filtration. The enzyme was produced by submerged fermentation of feather residue as a substrate. The purified collagenase showed a 2.09‐fold increase in specific activity and 22.85% yield. The enzyme was a monomeric protein with a molecular mass of 28.7 kDa, estimated by an SDS–PAGE and AKTA system. The optimum temperature and pH for enzyme activity were around 40°C and pH 8.0, respectively. The enzyme was strongly inhibited by phenyl‐methylsulfonyl fluoride, a serine protease inhibitor, and was thermostable until 65°C for 1 h. We then evaluated the enzyme's potential for degradation of Type I and Type V collagens for producing peptides with antifungal activity. Our results revealed that the cleavage of Type V collagen yielded more effective peptides than Type I, inhibiting growth of Aspergillus terreus , Aspergillus japonicus and Aspergillus parasiticus . Both groups of peptides (Type I and Type V) were identified by SDS–PAGE. To conclude, the thermostable collagenase we purified in this study has various potentially useful applications in the fields of biochemistry, biotechnology and biomedical sciences.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl₂, and CaCl₂, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl₂, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.     

Bioinspired synthesis,characterization and antifungal activity of enzyme-mediated gold nanoparticles using a fungal oxidoreductase

The development of efficient cell-free systems of nanoparticle synthesis using microbial enzymes is a growing field of biological and green chemistry for the supportable improvement in nano-biotechnology. In the present study, we established a cell-free system for producing gold nanoparticles (AuNPs) using a fungal oxidoreductase named sulfite oxidoreductase purified to homogeneity from Fusarium oxysporum. The enzyme was purified by ultrafiltration followed by anion exchange chromatography on DEAE Sephadex A-50 gel, and its molecular weight was determined by gel filtration chromatography on Sephacryl S-300 gel. The purified enzyme had a molecular weight of 346 kDa. It was composed of three subunits of 176, 94 and 76 kDa. Purified enzyme was successfully used for production of gold nanoparticles in a cell-free system. Synthesized gold nanoparticles showed the highest absorbance at 520 nm wavelength as shown by UV–visible spectroscopy. They were spherical in shape with an average size of 20 nm as determined by scanning and transmission electron microscopy and dynamic light scattering. Assessment of the antifungal properties of synthesized nanoparticles by disk diffusion method indicated a potent growth inhibitory activity against all tested human pathogenic yeasts and molds by inhibition zones ranged from 10 to 18 mm. Taken together, our enzymatically established method of nanoparticle synthesis using a purified sulfite oxidoreductase of F. oxysporum can be considered as an efficient tool for generating harmless bioactive gold nanoparticles with potential applications in biology, medicine and industry.     

Purification and characterization of Bacillus cereus protease suitable for detergent industry

An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.     

Purification and Characterization of Tyrosine Phenol Lyase from Citrobacter freundii

The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The K _m and V _max values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t _1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILE-TRP-TRP-VAL-GLY.     

A Novel Antifungal Protein with Lysozyme-Like Activity from Seeds of Clitoria ternatea

An antifungal protein with a molecular mass of 14.3 kDa was isolated from the seeds of butterfly pea (Clitoria ternatea) and designated as Ct protein. The antifungal protein was purified using different methods including ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-50 column. Ct protein formed a single colourless rod-shaped crystal by hanging drop method after 7 days of sample loading. The protein showed lytic activity against Micrococcus luteus and broad-spectrum, fungicidal activity, particularly against the most clinically relevant yeasts, such as Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus laurentii, Candida albicans and Candida parapsilosis. It also exerted an inhibitory activity on mycelial growth in several mould species including Curvularia sp., Alternaria sp., Cladosporium sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp., and Sclerotium sp. The present study adds to the literature on novel seed proteins with antifungal activity.     

Purification and Side Chain Selective Chemical Modifications of Glutamate Dehydrogenase from Bacillus subtilis Natto

Glutamate dehydrogenase (GDH) from Bacillus subtilis natto was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, size exclusion chromatography, and hydroxyapatite (HA) affinity chromatography. The GDH was purified 34-fold, with a yield of 41 % of total activity and a specific activity of 34.29 U/mg proteins. The molecular weight (M_r) of was measured at 47 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 264 kDa with high-performance liquid chromatography (HPLC). The optimum pH and temperature for the deammoniation reaction were measured to be 7.5 and 30 °C, respectively. The active-site amino acid residues of GDH were investigated by chemical modification. The compounds 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylglyoxal (PG), and phenylmethanesulfonyl fluoride (PMSF) were used to modify lysine, arginine, and serine active site residues, respectively. After treatment with modifying reagents at concentrations of 1 mM, GDH activity fell to 10.7 % with TNBS, 83.3 % with PG, and 12.8 % with PMSF. However, with substrate protection, there was almost no loss in GDH activity following treatment with any modifying reagent. The kinetic parameters K _m and V _max were determined in each case. K _m values for native GDH, 50 % TNBS-inactivated GDH, and 50 % PMSF-inactivated GDH were 0.037, 0.104, and 0.017 mM, respectively. V _max values were 0.048, 0.022, and 0.031 mM/s, respectively. These results suggest that the active site contains one or more lysine residues that play a role in substrate binding and one or more serine residues that may maintain the enzyme conformation. However, arginine residues played less of a role in the activity of GDH.     

Synthesis,characterization, antimicrobial evaluation and QSAR studies of organotin(IV) complexes of Schiff base ligands of 2-amino-6-substituted benzothiazole derivatives

A series of organotin(IV) complexes of type R₂SnLCl [R = Ph, Bu, Et, Me] were prepared by reaction of diorganotindichloride(IV) with Schiff base ligands, L₁ = (1-[(6-ethoxy-benzothiazol-2-ylimino)-methyl]-naphthalen-2-ol), L₂ = (1-[(6-nitro-benzothiazol-2-ylimino)-methyl]-naphthalen-2-ol), L₃ = (1-[(6-methoxy-benzothiazol-2-ylimino)-methyl]-naphthalen-2-ol) and L₄ = (1-[(6-methyl-benzothiazol-2-ylimino)-methyl]-naphthalen-2-ol) obtained from 2-amino-6-substituted benzothiazole derivatives with 2-hydroxy-1-naphthaldehyde in 1:1 molar ratio. These organotin(IV) complexes were characterized by various spectroscopic techniques (¹H, ¹³C and ¹¹⁹Sn NMR, FT-IR), and physical techniques (X-ray powder diffraction analysis and elemental analysis). The coordination of the prepared complexes has been planned as pentacoordinated around the central tin atom during which ligands coordinated to tin atom in bidentate manner acted as N, O donor system. The ligands and their complexes were screened for antibacterial and antifungal activities against Gram-positive bacteria Bacillus cereus (MTCC 10072), Staphylococcus aureus (NCIM 2901), Gram-negative bacteria Escherichia coli (MTCC 732), Pseudomonas aeruginosa (MTCC 424) and fungi Aspergillus niger (MTCC 9933) and Aspergillus flavus (ATCC 76801). The output of QSAR analysis indicated that topological parameters (molecular connectivity indices) were responsible for controlling the antimicrobial activity of the synthesized compounds.