农杆菌介导海洋草酸青霉转化体系及聚酮合酶Pks生物学功能*

为研究南海柳珊瑚共附生草酸青霉SCSGAF0023的聚酮合酶(PKS)生物学功能,采用农杆菌介导法构建草酸青霉SCSGAF0023的Pks敲除株ΔPks,比较野生菌株及ΔPks的生长发育及环境适应性差异。以草酸青霉SCSGAF0023分生孢子为受体,p0380-hygB为双元载体,成功实现草酸青霉SCSGAF0023的遗传转化。结果表明:农杆菌浓度为OD₆₀₀=0.5,在200μmol/L 乙酰丁香酮(AS)诱导下与10⁷个/ml草酸青霉SCSGAF0023孢子于25℃共孵育时转化效率最高。基于上述转化体系,成功获得Pks敲除株ΔPks,并首次证实Pks正向调控草酸青霉SCSGAF0023产孢,但不影响其对环境的适应性。这为进一步系统研究真菌PKSs及聚酮化合物对真菌生长发育与环境适应性的影响提供素材。     

草酸青霉YTY及其突变体解磷能力、pH和分泌有机酸的关系

本研究利用离子束-UV复合诱变草酸青霉YTY选育高效解磷突变体,分析了出发菌株YTY及其突变体解磷过程中的解磷能力、pH和有机酸的变化及其相关性,探讨草酸青霉的解磷机理。结果表明: 离子束-UV复合诱变选育获得5株高效解磷突变株P9-8、P9-9、P15-4、P15-6和P15-7,解磷能力均较YTY提高60%以上。解磷过程中突变株解磷能力及解磷速率均高于YTY,而pH显著低于YTY,同时,突变体分泌有机酸种类及含量发生了不同程度的变化,突变体和YTY均可分泌乳酸、乙酸和草酸,P9-8还产生了柠檬酸。皮尔逊相关分析显示,YTY及5株突变株的解磷能力和pH值呈显著负相关;YTY及其突变体(P15-4除外)的解磷能力与有机酸浓度和pH呈显著相关。分泌有机酸和降低环境pH值可能是草酸青霉解磷的内在机制,离子束-UV复合诱变可引起草酸青霉YTY有机酸分泌种类和分泌量的变化,还可能诱发YTY启动其他H⁺释放途径降低pH参与解磷。本研究为高效解磷青霉的开发及青霉解磷机理阐明提供了生物材料及理论依据。     

Purification and characterization of two novel β-glucosidases from Penicillium oxalicum and their application in bioactive ginsenoside production

Abstract

The fungus Penicillium oxalicum is able to selectively metabolize the 20(S)-protopanaxadiol ginsenosides Rb1, Rb2 and Rc to the bioactive ginsenoside compound K using extracellular glycosidases. In this study, two novel extracellular ginsenoside-hydrolyzing enzymes GH3-1 and GH3-2 were purified and characterized from P. oxalicum culture. Using ginsenosides as substrates, GH3-1 and GH3-2 synergistically catalyzed the hydrolysis of Rb1, Rb2 and Rc to yield the final product Compound K (C-K). The hydrolysis pathways were determined to be: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K and Rc→Mb→Mc→C-K for GH3-1 and GH3-2, respectively. The two enzymes differ, especially in composition, molecular weight, stability and substrate specificity, from GH1, a glycosidase previously purified from the same fungus. These enzymes could be of interest in glycoside degradation, especially in the production of minor ginsenosides.     

KINETIC STUDIES OF L-ASPARAGINASE FROM Penicillium digitatum

L-Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and other related malignancies. Its further use includes reduction of asparagine concentration in food products, which may lead to formation of acrylamide. Currently bacterial asparaginase is produced at industrial scale, but the enzyme isolated from bacterial origin is often associated with adverse reactions. These side effects require development of asparaginase from alternative sources. In the present study, Penicillium digitatum was explored for the production of extracellular L-asparaginase using modified Czapek–Dox media. The enzyme was purified about 60.95-fold and then kinetic study showed that the Km value of the enzyme was 1 × 10^?5 M. The optimum pH and temperature for the enzyme were 7.0 and 30°C, respectively. The optimum incubation period for L-asparaginase was 15 min. This work concludes that this enzyme can be a suitable candidate due to its strong kinetic properties, and further research can usher into development of asparaginase formulation from fungal origin with less adverse effects.     

Antifungal effect of Penicillium metabolites against some fungi

Microorganisms are increasingly exploited as a source of new biological control agents. Genus Penicillium is a source of novel bioactive molecules which can be used as antifungal agents. The objective of this study was to evaluate the antifungal potential of Penicillium strains. Culture filtrates of two Penicillium species were tested for their antifungal potential by well diffusion assays. Filtrate of Penicillium isolates showed high antifungal effects on mycelial growth of Fusarium oxysporum, Fusarium solani, Macrophomina phaseolina, Aspergillus japonicus var aculeatus and Cladosporium cladosporioides. But Penicillium italicum inhibit the fungal growth from 45 to 68% as compared to Penicillium simplissimum (25–68%). However in case of A. japonicus var aculeatus, Penicillium spp. extracts were equally effective and reduce the colony growth up to 68%. However, P. simplissimum extract was least effective in case of M. phaseolina, where it decreased the colony growth only 25%.     

Biocontrol of blue mold of apple by Candida membranifaciens in combination with silicon

In this study, antagonistic yeast Candida membranifaciens was combined with different concentrations of silicon (Si; 0, 0.1, 0.3 and 0.5% wt/vol) to evaluate the control of blue mold of apple in storage at 20°C and 5°C. Preliminary studies showed that Si at 0.6% or above inhibited mycelial growth of pathogens significantly in vitro. In vitro studies showed that Si at 0.1% had lower effect on yeast growth. In vivo studies showed that combination of different concentrations of Si with C. membranifaciens improved the efficacy of yeast in control of disease better than Si and yeast alone (P < 0.05). Our result showed that the effective concentration of Si is varied based on pathogen isolates and temperature, so that the most effective concentration of Si was 0.5% for isolate P2 at 20°C and 0.5% and 0.1% for isolates P1 and P2 at 5°C.     

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.