灰树花菌株的复壮及常压室温等离子体诱变

【背景】实验室所用灰树花菌株系长期继代培养,易出现菌株退化。【目的】通过菌株复壮的方法实现菌株的生物学活性及性状的恢复,并借助高效诱变仪对菌株实施诱变,以期得到活性更高、遗传稳定的诱变株。【方法】分别以PDA加富培养基和PDA-板栗壳培养基为培养基质,采用尖端菌丝分离法进行菌株复壮,得到回复菌株原有的生物学活性及性状的复壮株P-2,为了进一步提高菌株的高产性能,利用常压室温等离子体(atmosphericroomtemperatureplasma,ARTP)诱变技术作用于复壮株P-2菌丝体,最终筛选到一株性能优良、遗传稳定性高的诱变株b-35。【结果】复壮后的菌株P-2菌丝干重和多糖含量分别达到1.18%和19.01%,较出发株分别提高35.17%和35.11%,通过发酵罐验证菌株的发酵周期由48h缩短至32h,菌株发酵活性及效率明显提高。诱变株b-35菌丝干重和多糖含量分别达到1.56%和25.07%,较复壮株P-2分别提高了40.15%和39.33%。【结论】ARTP诱变方法易操作、无污染且诱变效率高,是获得灰树花高产菌株的重要方式。     

常压室温等离子体诱变选育微生物絮凝剂高产菌株

目的:利用常压室温等离子体诱变技术诱变海洋地衣芽孢杆菌DHS-40,选育微生物絮凝剂高产菌株。方法:10μL发酵液(D600nm值为0.5~0.7),辐射距离为2 mm,气流速度为10.0 SLPM,辐射功率为100 W。结果与讨论:辐射时间60~240 s为菌株DHS-40的最佳诱变照射时间,共分离得到396株诱变株;经过初筛、复筛,共计筛选得到8株絮凝活性较高的诱变菌;经DHS-40突变菌株与原始菌株同步培养,比较生长状态与絮凝活性,获得2株絮凝活性较高的突变菌株90-102与120-24,同期絮凝活性优于原始菌株。     

基于常压室温等离子体技术的金藻诱变筛选方法

以高生长速率或高油脂含量藻株为目标,以湛江等鞭金藻为例,报道了一种基于常压室温等离子体技术的微藻诱变及快速分级筛选方法。即以叶绿素荧光动力学参数F_v/F_m大于0.68的金藻为出发藻株、以致死率90%为阈值确定最适诱变电流为1.4~1.5A,诱变时间为24~30s。分别在室温常光及胁迫条件(高温常光和高光室温)下,按照孔板至摇瓶至反应器三级培养进行筛选。在孔板培养过程中以比生长速度结合尼罗红荧光强度变化实现高通量初筛,最终在反应器培养中进行提取验证,并以候选藻株为出发进行二次诱变筛选。结果表明,室温常光条件诱变株筛出率为0.7%,胁迫条件筛出率为0.9%;一次诱变诱变株筛出率为0.6%,二次诱变筛出率为1.2%;二次诱变和胁迫条件筛出率更高,更易得到性状变化的诱变株。     

常压室温等离子体诱变高效利用木糖产丁二酸菌株

大肠杆菌Escherichia coli AFP111是E. coli NZN111 (△pflAB△ldhA) 的ptsG自发突变株,其转化1 mol的木糖合成丁二酸的过程中净产生1.67 mol ATP,但是转化1 mol的木糖合成丁二酸的过程中实际需要2.67 mol ATP,因此在厌氧条件下,ATP的供给不足导致E. coli AFP111不能代谢木糖。采用常压室温等离子体射流诱变产丁二酸大肠杆菌菌株,在厌氧条件下,利用以木糖为碳源的M9培养基,筛选得到一株可以代谢木糖并积累丁二酸的突变株DC111。该突变菌株在发酵培养基中,72 h内可以消耗10.52 g/L木糖产6.46 g/L的丁二酸,丁二酸的得率达到了0.78 mol/mol。而且突变株中伴有ATP产生的磷酸烯醇式丙酮酸羧激酶 (PCK) 途径得到加强,PCK的比酶活相对于出发菌株提高了19.33倍,使得其在厌氧条件下能够有足够的ATP供给来代谢木糖发酵产丁二酸。     

常压室温等离子体技术在微生物诱变中的应用进展

微生物是人类赖以生存的重要资源,为提高微生物的生产效率或者赋予其新的生物学功能,需要通过理化方法进行诱变或通过分子生物学技术对其进行定点突变。在目前的理化诱变方法中,常压室温等离子（atmospheric and room temperature plasma,ARTP）诱变技术具有操作简单、条件温和、安全性高、诱变快速等优点,成为倍受青睐的新方法。基于此,综述了ARTP诱变技术的原理及其在微生物诱变育种方面的应用,以期为选育性能优越的微生物菌种的诱变育种相关研究提供借鉴。     

常压室温等离子体诱变选育花脸香蘑新菌株

花脸香蘑作为一种营养丰富、菇型美观的珍稀食用菌,具有良好的开发利用价值.利用常压室温等离子体(ARTP)技术对野生花脸香蘑菌株610双核菌丝片段进行了诱变处理.利用拮抗试验进行了诱变菌株初筛.通过连续传代培养,自然淘汰劣势菌株并获得诱变效应稳定的48个菌株.综合平板培养和基于ISSR和SSR联合遗传多样性分析的结果,筛...     

常压室温等离子体诱变选育类胡萝卜素高产菌株及其性质研究

采用新型常压室温等离子体(Atmospheric and room temperature plasma,ARTP)诱变产类胡萝卜素菌株Deinococcus wulumuqiensis R12,通过深孔培养板进行高通量筛选,并结合菌株类胡萝卜素含量复筛获得突变菌株M1。与出发菌株相比,突变菌株M1的类胡萝卜素含量检测为612μg/g细胞干重(Dry cell weight,DCW),是出发菌株类胡萝卜素含量(212μg/g DCW)的2.8倍,且遗传性稳定;突变菌株M1类胡萝卜素的高铁离子还原能力(OD700=0.52)高于出发菌株类胡萝卜素的高铁离子还原能力(OD700=0.34),且M1的类胡萝卜素对DPPH自由基清除率为33.33%高于出发菌株类胡萝卜素的清除率(23.09%),结果显示突变菌株M1具有更强的抗氧化能力;在相同γ辐射与UV辐射剂量下突变菌株M1具有比出发菌株更高的存活率。研究表明突变菌株M1在类胡萝卜素产量、菌株抗氧化性能及抗辐射性能方面均优于出发菌株D.wulumuqiensis R12。     

基于核糖体工程理论的常压室温等离子体诱变筛选多杀菌素高产菌

利用核糖体工程理论,采用常压室温等离子体(ARTP)诱变对刺糖多孢菌(Saccharopolyspora spinosa)进行诱变筛选。以刺糖多孢菌QYLZ 88912菌株为出发菌株,对其进行ARTP诱变,选取三个不同致死率的照射时间,将孢子悬液混合,以增加抗性筛选几率。然后基于核糖体工程技术理论对混合孢子悬液进行初筛,最终筛选出了1株多杀菌素高产菌Sg200Rif110St40Er90-028。该菌株同时具有磺胺胍、利福平、链霉素、红霉素的多重抗性,摇瓶发酵试验表明,发酵7d后多杀菌素浓度可达到1 516.93 mg/L,较出发菌株QYLZ 88912的产量610.75 mg/L提高了148.37%,且遗传性稳定。以得到的高产菌Sg200Rif110St40Er90-028作为出发菌株,对发酵培养基进行响应面优化实验,优化后的培养基(g/L):葡萄糖46.97、麦芽糊精35、酵母粉40.36、水浸棉籽粉32.88、碳酸钙3,多杀菌素的产量为2 361.81 mg/L,比出发菌株的产量提高了286.71%。     

阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

雷帕霉素菌株的常压室温等离子体(ARTP)诱变选育

雷帕霉素由放线菌次级代谢产生,具有抗真菌、抗肿瘤以及免疫抑制等生理活性,是临床重要的器官移植、抗肿瘤原料药物。为了获得具有高发酵潜力的工业用菌株,对雷帕霉素菌株的摇瓶发酵工艺进行了优化。采用常压室温等离子体(ARTP)的方法对高产雷帕霉素菌株的单孢子悬液进行诱变处理,通过高压液相的方法对诱变菌株的发酵代谢产物进行检测,成功地从诱变菌株中筛选获得了发酵产量提高30%以上的诱变菌株,建立了ARTP诱变选育高产雷帕霉素菌株的方法,为后期进行该菌株的选育研究工作以及中试发酵工艺优化提供了方法和物质的基础。     

常压室温等离子体(ARTP)诱变茂源链霉菌菌株

采用常压室温等离子体（ARTP）诱变技术处理茂源链霉菌（Streptoverticillium mobaraense）菌株HS47的孢子,选育微生物谷氨酰胺转胺酶（MTG）高产菌株。菌株的致死率强度和正突变率高低结果表明,在电源功率为120W,处理距离2mm,工作气流量10slpm时,等离子体氦气对茂源链霉菌HS47孢子的最佳处理时间为30s。将诱变后的孢子液稀释涂布后,利用96孔板高通量筛选方法对单菌落进行初筛,选出高产的突变株进行两轮试管复筛,筛选过程中保持对菌株的分离纯化,最终获得一株高产菌株M-8,其MTG酶活由2.8U/ml提高到5.1U/ml,较出发菌株HS47提高了82%。该菌株的摇瓶发酵实验证明,其酶活的提高是单位菌株分泌的MTG有所增加的结果。经过8次传代,证实该菌株具有良好的遗传稳定性。这为谷氨酰胺转胺酶的工业化生产提供了菌种支持和理论支持。     

Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9^th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.     

1株解淀粉芽胞杆菌的常压室温等离子诱变及其发酵优化

为提高解淀粉芽胞杆菌(Bacillus amyloliquefaciens)TXM-A2对镰刀菌的抑菌作用,利用常压室温等离子(Atmospheric Room Temperature Plasma,ARTP)诱变技术处理菌株TXM-A2,以禾谷镰孢菌(Fusarium gra-minearum)为筛选指示菌,获得1株...     

Atmospheric Pressure Room Temperature Plasma Jets Facilitate Oxidative and Nitrative Stress and Lead to Endoplasmic Reticulum Stress Dependent Apoptosis in HepG2 Cells

Atmospheric pressure room temperature plasma jets (APRTP-Js) that can emit a mixture of different active species have recently found entry in various medical applications. Apoptosis is a key event in APRTP-Js-induced cellular toxicity, but the exact biological mechanisms underlying remain elusive. Here, we explored the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in APRTP-Js-induced apoptosis using in vitro model of HepG2 cells. We found that APRTP-Js facilitated the accumulation of ROS and RNS in cells, which resulted in the compromised cellular antioxidant defense system, as evidenced by the inactivation of cellular antioxidants including glutathione (GSH), superoxide dismutase (SOD) and catalase. Nitrotyrosine and protein carbonyl content analysis indicated that APRTP-Js treatment caused nitrative and oxidative injury of cells. Meanwhile, intracellular calcium homeostasis was disturbed along with the alteration in the expressions of GRP78, CHOP and pro-caspase12. These effects accumulated and eventually culminated into the cellular dysfunction and endoplasmic reticulum stress (ER stress)-mediated apoptosis. The apoptosis could be markedly attenuated by N-acetylcysteine (NAC, a free radical scavenger), which confirmed the involvement of oxidative and nitrative stress in the process leading to HepG2 cell apoptosis by APRTP-Js treatment.     

常压室温等离子体(ARTP)诱变快速选育高产DHA的裂殖壶菌突变株

旨在建立一种能够快速便捷的诱变选育高产DHA菌株的方法。出发菌株Schizochytrium sp.ATCC 20888悬浮液经过常压室温等离子体(ARTP)处理后,涂布到2,2’-联吡啶平上板培养。将所得的突变菌株摇瓶发酵培养,通过磷酸香草醛油脂快速检测法和气相色谱分析从突变菌株中筛选得到DHA高产菌株。结果表明,裂殖壶菌诱变选育条件为ARTP为处理时间15 s,气量10 L/min,电功率100 W;2,2’-联吡啶浓度为100μmol/L。通过该方法可以获得高产DHA的菌株。其中D32菌株DHA生产能力提升显著,比初始菌株提升了29.8%,DHA产量达到7.31g/L。D32菌株与出发菌株相比,主要的饱和脂肪酸含量显著下降(P0.005),而不饱和脂肪酸含量显著增加(P0.005)。经5次传代后性状稳定,本方法快捷高效,同时也为其他多不饱和脂肪的诱变选育方法提供参考。     

物化因子结合诱变选育耐温型法夫酵母

以法夫酵母为出发菌株,采用紫外线及甲基磺酸乙酯对其进行诱变育种,筛选出1株最适生长温度比诱变前提高10℃且虾青素产量可达到5．08mg／L的突变菌株,该菌株经多次传代生产性能稳定。     

Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (～10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.