Biological properties of phospholipase C purified from a fleecerot isolate of Pseudomonas aeruginosa

A role for one of many exocellular enzymes produced by Pseudomonas aeruginosa--phospholipase C (PLC)--as a prime candidate virulence factor in fleecerot dermatitis has been examined. The addition of Tween 80 in tryptose minimal medium effectively perturbed the membrane system of a field isolate of P. aeruginosa, resulting in increased production and release of a periplasmic enzyme marker, alkaline phosphatase (AP), and also of PLC. PLC activity levels in the culture supernatant were 10- to 15-fold higher in the presence of Tween than in its absence. Apart from AP, the culture medium contained little or no detectable proteolytic enzyme activity, thereby facilitating the partial purification of a haemolytic form of PLC by anion-exchange chromatography. This enzyme, when injected intradermally into the skin of sheep, elicited histopathological lesions virtually identical to those seen in naturally occurring fleecerot. In addition, serum from each of eight sheep afflicted with fleecerot contained high levels of circulating anti-PLC antibody activity when assayed by ELISA. Since these antibodies did not affect the enzymic function of PLC, it is likely that they do not bind to, or are incapable of conformational modification of, the active site.     

人羊水祖细胞的分离和基因修饰

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果,为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落,经培养传代后,通过第3代慢病毒载体将hFIX导入该细胞,经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样,倍增时间为39.05 h,该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4,TRA1-60,在基因水平还可检测到NANOG,OCT4,SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后,能合成并分泌hFIX蛋白,传代后48 h在上清液中的浓度为20.37%±2.77%,凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期,为50.35%±5.42%,凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势,波动幅度较小;同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞,转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白,为血友病B产前治疗的新方法提供了实验依据。     

Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099

This study reports the production of xylanolytic and cellulolytic enzymes by a thermophilic fungal isolate Myceliophthora sp. using a cheap medium containing rice straw and chemically defined basal medium under solid-state culture. A combination of one factor at a time approach followed by response surface methodology using Box-Behnken design of experiments resulted in 2.5, 1.25, 1.28 and 4.23 fold increase in xylanase, endoglucanase, beta-glucosidase and FPase activity, respectively. The zymograms developed against IEF gels showed that multiple isoforms of xylanase (5), endoglucanase (4) and beta-glucosidase (2) were produced under optimized culture conditions. Moreover, thiol containing serine proteases produced during the growth of the culture had no role in the post-translational modification of these xylanases.     

Influence of carbon source on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from wheat

The fungus Pyrenophora tritici-repentis can infect wheat kernels, causing red smudge, and has been shown to produce the anthraquinone mycotoxins emodin, catenarin, and islandicin. The growth of 8 fungal isolates from diverse regions was evaluated on various culture media and was found to be generally slowest on the semisynthetic Fries medium. The choice of carbon source had a significant effect on mycotoxin production, as assessed by high-performance liquid chromatography. The highest emodin concentration (194.18 ± 16.26 μg/g medium) was observed for isolate Alg 3-24 on Fries medium supplemented with fructose, while the highest catenarin concentration (302.54 ±13.92 μg/g medium) was observed for TS93-71B on Fries medium supplemented with starch. Islandicin was not produced by any isolate under the conditions tested. Evaluation of the dynamics of mycotoxin production by isolate 331-2 on V8-potato dextrose agar medium revealed a rapid accumulation of emodin and catenarin during the first week of incubation, followed by a large decline by 14 days. Differences in the growth of and mycotoxin production by isolates of P. tritici-repentis likely resulted from the differential composition of the media and (or) intraspecies variability. Accordingly, the optimization of growth medium should be considered when evaluating the potential of specific isolates for mycotoxin production.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Characterization of the cellulolytic activity of a Bacillus isolate

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.     

Production of (R)-1-(4-Bromo-phenyl)-ethanol by locally isolated Aspergillus niger using ram horn peptone

Aspergillus niger EBK-9 was isolated from soil sample. This isolate was evaluated for production of (R)-1-(4-Bromo-phenyl)-ethanol 2 from 1-(4-Bromo-phenyl)-ethanone 1. In this work, the production of the 2 was achieved via fermenter. Glucose, yeast extract and ram horn peptone as medium in fermenter for growth of A. niger was used. A. niger EBK-9 isolate was found to be an effective biocatalyst with excellent enantiomeric excess (>99%) and good conversion (100%) for the production of the 2 in batch culture. The 8.2 mmol/l product from 10 mmol substrate under the optimum conditions could be produced. The yield was calculated as 82%. Because of the easy availability of the fungus besides simple reaction conditions, this process and medium must be potentially useful for production of chiral alcohols.     

链孢粘帚霉几丁质酶的诱导及其抗真菌活性研究

链孢粘帚霉HL-1-1菌株在不同培养条件下产几丁质酶活性不同,核盘菌菌核对其几丁质酶的诱导作用高于几丁质;首次用菌核配制培养基成功诱导了几丁质酶的产生,其产酶的最适初始pH值为4.5,最适培养时间为6d。几丁质酶对10种病原菌都有不同的抑制作用,对小麦雪腐病、葡萄白腐病、玉米黄斑病及斑点落叶病等病原菌的孢子萌发具有明显的抑制作用;该几丁质酶还能明显抑制核盘菌和立枯丝核菌的菌核萌发。     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.