Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta,Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37?U/ml and 98.25?U/ml, respectively, after 72?h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35?°C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45?°C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72?kDa.     

In vitro fibrinolytic activity of an enzyme purified from Bacillus amyloliquefaciens strain KJ10 isolated from soybean paste

A fibrinolytic protease secreting producing Bacillus amyloliquefaciens strain KJ10 was initially screened from the fermented soybean. Maximum productivity was obtained in the culture medium after 40 h incubation, 34 °C incubation temperature at pH 8.0. Fibrinolytic protease production was enhanced in the culture medium with 1% sucrose (3712 ± 52 U/mL), 1% (w/v) yeast extract (3940 ± 28 U/mL) and 0.1% MgSO₄ (3687 ± 38 U/mL). Enzyme was purified up to 22.9-fold with 26%recovery after Q-Sepharose HP column chromatography. After three steps purification, enzyme activity was 1606U/mg and SDS-PAGE analysis revealed 29 kDa protein and enzyme band was detected by zymograpy. Enzyme was highly active at pH 8.0, at wide temperature ranges (40 °C ? 55 °C) and was activated by Mn²⁺ (102 ± 3.1%) and Mg²⁺ (101.4 ± 2.9%) ions. The purified fibrinolytic enzyme was highly specific against N-Suc-Ala-Ala-Pro-Phe-pNA (189 mmol/min/mL) and clot lytic activity reached 28 ± 1.8% within 60 minin vitro. The purified fibrinolytic enzyme showed least erythrocytic lysis activity confirmed safety to prevent various health risks, including hemolytic anemia. Based on this study, administration of fibrinolytic enzyme from B. amyloliquefaciens strain KJ10 is safe for clinical applications.     

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8?U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39?kDa. Optimal activity of the enzyme was observed at 50?°C and pH 10. The enzyme exhibited stability up to 60?°C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.     

Production of a fibrinolytic enzyme by thermophilic Streptomyces species

A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent culture broth of a thermophilic organism, Streptomyces megasporus SD5. The strain could produce 150 mg crude protein per litre of spent broth, with a specific activity of 80 IU (Plough units) per milligram, within 18 h of incubation at 55 °C in glucose yeast/extract/peptone (GYP) medium, pH 8.0. For production of the enzyme, the strain could utilize different carbon and nitrogen sources with a C:N ratio of ∼ 1:2. The enzyme was stable at a broad range of pH ranging from 5 to 9, and highly thermostable with 50% activity after storage at 60 °C for 6 months. The enzyme belonged to the serine endopeptidase group. In vitro clot lysis revealed that the enzyme was active at 37 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Study of some parameters which affect xylanase production: Strain selection, enzyme extraction optimization, and influence of drying conditions

Xylanases are glycosidases mainly responsible for the hydrolysis of β-1,4 linkages in xylan. Xylanase was produced in this work by solid-state fermentation using agro industrial residues with Aspergillus niger strain, which was screened through qualitative and quantitative methods. Extraction processes with different solvents were evaluated. Solvent volume, time, and agitation speed (shaker) were optimized using statistical designs. Drying studies of the solid fermented material were also conducted in a laboratory oven where the following conditions were applied: 42°C and 50°C for 20 h and 80°C for 1 h; 50°C and 75°C for 6 and 3 h, respectively. Best extraction conditions were 37 mL of solvent composed by NaCl solution (0.9%) with Tween 80 (0.1%) in 3 g of cultured material with agitation at 133 rpm in shaker for 4 min. Highest xylanase activity was 2,327 IU/gdm. The drying at 42°C for 20 h provided a better maintenance of xylanase activity (58% of xylanase activity).     

Bactericidal Activity of a Fermented Hot-Water Extract from Stevia rebaudiana Bertoni towards Enterohemorrhagic Escherichia coli O157:H7 and Other Food-Borne Pathogenic Bacteria

A fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157:H7. The colony-forming ability of the food-borne pathogenic bacteria tested so far was reduced to < 10^?7 when exposed to ?40% (v/v) solutions of the fermented extract at 37 C for 2 hr. Secretion of verocytotoxin 1 and 2 by enterohemorrhagic E. coli was also diminished by fermented extract at a concentration of ?10% (v/v). In contrast, the fermented extract did not significantly kill Bifidobacteria or Lactobacilli. The active principle(s) of the fermented Stevia extract were bactericidal under acidic conditions.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

一株产纤溶酶菌株的分离、鉴定及其酶活特征的初步研究

[目的]分离筛选并鉴定产纤溶酶的菌株.[方法]采用血粉培养基富集,琼脂糖-纤维蛋白平板筛选,从自然界中分离筛选出一株产纤溶活性物质的菌株.通过形态学特征、生理生化特征研究,并结合16S rRNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类.[结果]从自然界分离筛到一株产纤溶酶的菌株EF608,经鉴定该菌株为粪肠球菌(Enterococcus faecalis). SDS-PAGE和纤维蛋白自显影表明该纤溶酶的分子量为37 kD,最适反应温度和pH分别为35℃和7.5,EDTA能完全抑制其纤溶活性,而PMSF对其活性无抑制作用.菌株EF608发酵液不仅可以直接水解纤维蛋白,而且具有体外溶栓的作用,对血红细胞没有溶解作用.[结论]筛选到一株具有纤溶活性的粪肠球菌——EF608,为获取新型纤溶酶提供了一种的新的菌源.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

Production,purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21?U/mL, which was ～2.3-fold higher than that of the wild strain (2.04?U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32?°C, pH 7.0, 150?rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25?mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30?kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ～21-fold, and the mean overall yield was ～6%. The purified endoglucanase was active up to 80?°C and showed a half-life of 214?min at 60?°C in the absence of substrate at pH 8.0. The apparent K_m value for the purified endoglucanase was 0.70?mg/mL, while the V_max value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30?U of proteinase K/mg. The addition of Mg⁺² and Ca⁺² (5?mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5?mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4?°C and 75 days at ?20?°C signify the suitability of this enzyme for industrial applications as detergent additive.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0?kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′ tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.     

中华婪步甲共生放线菌的抗菌活性筛选及菌株BJ37的初步鉴定

利用平板分离法从中华婪步甲(Harpalus sinicus)中分离得到56株共生放线菌。其中菌株BJ37具有较好的抗菌活性,其发酵液对枯草芽胞杆菌和白色念珠菌的抑菌圈直径均大于30.0 mm。进一步研究BJ37发酵液不同极性溶剂萃取物的抗菌活性,结果表明在中等极性部位存在活性物质。通过形态学特征和分子生物学分析确定该菌株相似菌株为委内瑞拉链霉菌(Streptomyces venezuelae)。菌株BJ37作为微生物杀菌剂潜在产生菌值得进一步研究。     

Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ～33.76?kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bβ chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35?°C, pH 8. Ca²⁺ stimulated the fibrinolytic activity of xylarinase while Fe²⁺ and Zn²⁺ inhibited suggesting it to be a metalloprotease. The K_m and V_max values of xylarinase were 240.9?μM and 1.10?U/ml for fibrinogen and 246?μM and 1.22?U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.     

Optimization of the fermentation parameters for the production of Ganoderma lucidum immunomodulatory protein by Pichia pastoris

Abstract

In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L₉(3³) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5?L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71?μg/ml in the shake-flask, and 613.47?μg/ml in the 5?L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280?rpm for 4?days at 26?°C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

The male gametophyte of Bonnemaisonia hamifera Hariot in Norway

The male gametophyte of Bonnemaisonia hamifera Hariot is recorded for the first time in Norway. The plants were collected in the Bj?r?ya archipelago situated between 10° 46′-10° 52′ E and 64° 35·5′-64° 37′ N. Numerous large antheridia were present on the plants which were attached to the rock at a depth of 150–200 cm below E.L.W.S.     

Optimization of extracellular lipase production from the biocontrol fungus Nomuraea rileyi

Lipases are important cuticle-degrading enzymes that hydrolyze the ester bonds of waxes, fats and lipoproteins during the infection of insects by the fungus Nomuraea rileyi. Lipase production by the N. rileyi strain MJ was optimized by varying environmental and nutritional conditions in culture medium containing different vegetable oils at various concentrations with shaking at 150 rpm for 8 days at 25°C. The maximum lipase production was obtained using castor oil (30.5±0.6 U mL^?1), followed in order by coconut oil (20.8±0.4 U mL^?1), olive oil (20.8±0.4 U mL^?1) and cottonseed oil (20.6±0.4 U mL^?1). The highest lipase activity (37.7±0.4 U mL^?1) was obtained when castor oil was used at a concentration of 4% (v/v) of basal medium. When the surfactant Tween 80 was added at the fourth day rather than at the beginning of incubation, a maximum lipase activity of 44.9±3.5 U mL^?1 was obtained. The optimal temperature and pH for lipase production were 25°C and pH 8.0, respectively. This is the first report on lipase production by the biocontrol fungus N. rileyi.     

Biochemical analysis of a fibrinolytic enzyme purified from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain A1

A fibrinolytic enzyme from Bacillus subtilis strain Al was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain Al was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0–10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain Al.