Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

氮源对阿维菌素合成的影响及基于二氧化碳释放速率的阿维菌素发酵调控

中国是世界上最大的也是唯一的阿维菌素原料生产国,但在工业规模生产中与同类型大环内酯类抗生素相比其产量相对偏低。文中通过研究不同氮源对阿维链霉菌生长、代谢的影响,发现氮源在发酵中后期对菌丝活性、菌丝浓度以及阿维菌素B1a的合成都有较为显著的影响。在100 L生物反应器中,于发酵中后期基于二氧化碳释放速率(CER)控制补入酵母粉,效价达到8697mg/L,与原工艺相比,提高了26.9%。这一结论若在实际工业生产中应用,有望带来实际的经济效益。     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml^–1), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml^–1) yielded 27 and 21 g ml^–1 oxytetracycline compared with 97 and 104 g ml^–1 for unsterilised gels (3.6 and 5.2 mg ml^–1 biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.     

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K_m and k_cat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min^?1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704. © 2009 Wiley Periodicals.     

三株杀粘虫放线菌的分类鉴定

菌株YIM31331、YIM31333、YIM31355是从云南省丽江、中甸采集的土壤样品中分离得到的具有产杀粘虫活性物质菌株。根据其形态学特征、生理生化特性及16S rDNA序列分析结果,认为菌株YIM 31331属于链霉菌属的Streptomyces subrutilus,YIM31333属于指孢囊菌属Dactylosporangium aurantiacum,YIM31355属于链孢囊菌属Streptosporangium vulgare。     

链霉菌基因转移的方法

本文介绍了用于链霉菌基因转移的各种方法 ,涉及原生质体转化、电穿孔、接合转移和噬菌体转导 ,对影响链霉菌基因转移的各种因素进行了分析。     

放线菌制剂对人参生长及根域土壤微生物区系的影响

以小兴安岭地区人参为研究对象,探索放线菌制剂对人参的促生效应及对人参根区、根表土壤微生物区系的影响.结果表明： 经放线菌制剂Streptomyces pactum（Act12）处理后,人参药用部分产量增加,品质改善;叶片诱导酶活性提高,根系活力增强;土壤中细菌、放线菌的数量和比例显著增加,真菌的数量和比例减少.与对照相比,土壤微生物区系结构改变:优势菌荧光假单胞菌、韩国假单胞菌和氧化微杆菌在根区、根表土壤中的数量大幅提高;病原真菌烟色织孢霉在根区土壤中减少,在根表土壤中消失.表明施用放线菌制剂Act12能够改善土壤微生物区系,提高人参植株的抗性和根系活力,增加产量并改善品质.     

Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp. F2621 isolated in Turkey

AIMS: To determine the effect of environmental conditions on the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and to assess the potential use of these enzymes in the hydrolysis of lignocellulose material. METHODS AND RESULTS: The production of extracellular lignocellulose-degrading enzymes, endoxylanase, endoglucanase and peroxidase during the growth of Streptomyces sp. F2621 in basal salts-yeast extract medium containing different carbon sources and the effect of a number of environmental parameters (e.g. carbon sources and concentrations, pH and temperature) were investigated. The highest endoxylanase (22.41 U ml(-1)) and peroxidase (0.58 U ml(-1)) activities were obtained after 2-4 days of incubation at 30 degrees C in a basal salts medium containing 0.4% (w/v) oat spelt xylan and 0.6% (w/v) yeast extract, corresponding to C : N ratio of 6 : 1. Cell-free extracellular enzyme preparations from the strain were capable of releasing both sugar and aromatic compounds during incubation with eucalyptus paper pulp, straw and xylan. Overall, 9.3% hydrolysis of xylan occurred after 24-h incubation. However the rates of hydrolysis of paper pulp and straw were approximately twofold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate (24.5% and 16.3%, respectively) was greater than xylan hydrolysis. CONCLUSIONS: The high levels of enzyme production achieved under batch cultivation conditions, coupled with no significant production of endoglucanase during the growth phase of organism and the release of both sugar and aromatic compounds from paper pulp and straw signify the suitability for these enzymes for industrial applications such as pulp and paper production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the environmental conditions for the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and suggest the use of streptomycetes and/or their enzymes in industrial processes.     

生防链霉菌ZM-16的发酵产物生物活性及其微波诱变育种

【目的】链霉菌ZM-16对多种细菌有良好的抑菌作用,其发酵产物的主要活性物质为放线菌素D。本研究旨在进一步探讨其抗真菌活性,并通过诱变育种的方法对菌种进行改良,以提高活性物质的产量,从而为其在植物病害生物防治领域的实践应用奠定基础。【方法】利用液体发酵的方法获取活性产物并对其进行粗提取;利用抑菌圈法检测菌株发酵产物对11种植物病原真菌的抑制作用;通过微波处理并利用抗生素抗性筛选的方法对菌株进行诱变育种。【结果】粗提液对11种植物病原真菌均具有抑菌效果,最为明显的3种病菌分别是苹果炭疽病菌、油菜菌核病菌和禾谷镰刀病菌;经微波诱变筛选到了一株耐利福平突变株,其放线菌素D的产量提高了36.75%;传代研究表明其经过10代选育,遗传稳定性较好。【结论】链霉菌ZM-16的发酵产物具有良好的抗植物病原真菌的活性,经过育种后其活性产物产量也有所提高,因此具有较高的实际应用价值。