“等离子体-紫外复合诱变选育四羟基环孢菌素衍生物高产菌株”主要仪器信息更正

<正>经本刊2014年第34卷第1期第一作者章丽对其文章"等离子体-紫外复合诱变选育四羟基环孢菌素衍生物高产菌株"中主要仪器信息确认,"常温常压等离子体射流诱变机"应为"常压室温等离子体诱     

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。     

常压室温等离子体诱变选育L-精氨酸生产菌及发酵条件优化

【目的】通过常压室温等离子体诱变技术选育L-精氨酸高产菌株,利用响应面设计探索突变菌株生产L-精氨酸的最佳发酵条件。【方法】采用常压室温等离子体生物诱变系统对实验室保藏的Corynebacterium glutamicum GUI089进行系列诱变,选育L-高精氨酸和8-氮鸟嘌呤抗性菌株。在单因子实验的基础上,应用Plackett-Burman设计从7个因素中筛选出对L-精氨酸合成具有显著效应的(NH4)2SO4、葡萄糖和尿素3个因素。基于上述结果,进一步采用响应面设计优化出主要影响因素的最佳参数水平。【结果】经过一系列的诱变和筛选,选育出一株L-高精氨酸(15 g/L)和8-氮鸟嘌呤(0.7 g/L)抗性菌株,并将此菌株命名为C.glutamicum ARG 3-16。此菌株的L-精氨酸产量比出发菌株提高了49.79%,且发酵液中杂酸的浓度明显降低,特别是L-脯氨酸、L-谷氨酸和L-缬氨酸。在经响应面优化后的最佳发酵条件下,L-精氨酸的产量达到39.72±0.75 g/L,比优化前提高了10.49%。【结论】通过常压室温等离子体诱变技术成功选育出一株L-精氨酸高产菌株,利用响应面法有效地优化了发酵条件,实验结果表明突变株ARG 3-16具有潜在的生产应用价值。     

等离子体-紫外复合诱变选育四羟基环孢菌素衍生物高产菌株

筛选可体内转化合成环孢菌素衍生物([γ-HyMeLeu4]CyA)的高产菌株。以稀有放线菌Nonomuraea dietziae为出发菌株,采用常温常压等离子体射流和紫外照射对其进行复合物理诱变。通过该诱变方法获得了具有稳定遗传性能的菌株,其转化效率比出发菌株提高了32%左右。结果表明,等离子体诱变可有效的用于该稀有放线菌,通过等离子体-紫外复合诱变筛选得到的突变菌株可用于工业生产。     

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。     

霉酚酸高产菌株短密青霉菌SA-2的选育

为选育出霉酚酸（mycophenolic acid, MPA）发酵生产的优良菌株,以产霉酚酸的短密青霉菌（Penicillium brevicompactum）S-225菌株作为原始菌株,利用甲基磺酸乙酯（ethyl methanesulfonate, EMS）、紫外诱变（ultraviolet, UV）和常压室温等离子体诱变（atmospheric room temperature plasma, ARTP）叠加诱变的方法,选育出1株遗传稳定、且对Cu²⁺耐受性强的菌株SA-2,其10 t中试霉酚酸发酵效价达到8 458 mg/L,比原始菌株提高了137.3%,且其200 t发酵生产效价达到了8 769 mg/L。该研究结果对霉酚酸工业化发酵生产具有重要意义。     

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。     

L-异亮氨酸高产菌选育及其培养基优化

旨在诱变选育L-异亮氨酸高产菌,并探索突变株最佳发酵条件。利用传统化学诱变结合常压室温等离子体生物诱变体系对实验室保藏的Brevibacterium flavum I-12进行逐级诱变,选育2-噻唑丙氨酸(2-TA)和磺胺胍(SG)高抗性和在琥珀酸平板上能快速生长的突变菌株。随后,在单因素实验的基础上,利用响应面设计优化出目的突变株摇瓶发酵培养基组分的最佳参数水平。结果显示,经过一系列诱变和筛选,成功选育出一株在40 g/L的2-TA和5 g/L的SG,且以琥珀酸为唯一碳源的培养基上快速生长突变株,命名为B. flavum TA-6,该菌株产酸达26.2±0.5 g/L,比出发菌株提高了44.75%,而副产物L-缬氨酸和L-亮氨酸积累量明显降低。经响应面法优化发酵条件后,突变株产酸可达27.8±0.5 g/L,比优化前提高了6.1%。通过传统化学诱变结合ARTP生物诱变体系,成功选育出一株杂酸降低的L-异亮氨酸高产菌TA-6,该菌株具有潜在生产应用价值。     

常压室温等离子体-紫外复合诱变选育β-法尼烯高产菌株

【背景】大肠杆菌(Escherichia coli)由于生长性能优良、遗传背景清楚、遗传操作手段成熟,是合成β-法尼烯的合适生产菌,但其合成β-法尼烯的产量目前仍不能满足工业化生产的需求。【目的】通过诱变筛选技术选育β-法尼烯高产突变株。【方法】采用常压室温等离子体(atmosphericand room temperature plasma,ARTP)诱变技术和紫外线照射对出发菌株大肠杆菌EC-16进行复合诱变,并以异戊烯焦磷酸耐受性为选择压力进行平板初筛,之后进行摇瓶复筛,最后进行发酵罐验证。通过连续多代培养筛选到的高产突变菌株,观察其遗传稳定性。【结果】经复合诱变选育筛选出一株β-法尼烯高产突变株E.coliHVK-9,其产量高达22.1g/L,相比出发菌株提高了168.74%。【结论】采用ARTP-紫外复合诱变,再结合异戊烯焦磷酸抗性筛选的集成方法,使得诱变菌株的正突变率大大提高,可以有效地提高诱变菌株的β-法尼烯产量。突变株HVK-9作为工业化发酵生产菌种具有较好的遗传稳定性,为β-法尼烯的工业化生产和应用奠定了良好的基础。     

常温常压等离子体诱变选育高产L-异亮氨酸大肠杆菌

旨在选育L-异亮氨酸高产大肠杆菌.以大肠杆菌K12(Met-)为出发菌株,经常温常压等离子体(ARTP)诱变,通过微生物高通量液滴培养系统(MMC)筛选,以α-氨基丁酸(α-AB)抗性为筛选标记,得到一株高产L-异亮氨酸的突变菌株大肠杆菌NXU12,并对其遗传稳定性进行了研究.结果表明,出发菌株大肠杆菌K12(Met-...     

植酸酶菌种筛选方法的研究

通过微板培养法对植酸酶产生菌菌种筛选方法进行了研究,建立了一种快速、有效的高通量植酸酶菌种筛选方法。利用该方法结合菌种诱变技术已选育出植酸酶酶活高达297u／mL的A,niger 496-1菌株。     

High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.     

Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett–Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO₄, KCl, incubation period, and MnSO₄ are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.     

卡氏德巴利酵母植酸酶在毕赤酵母中的异源表达及优化

【背景】植酸是一种能螯合金属离子和蛋白质的有机磷类化合物,广泛存在于植物组织中,影响动物对营养元素的吸收。在饲料中加入植酸酶可有效降解植酸。【目的】构建毕赤酵母异源表达卡氏德巴利酵母(Debaryomyces castellii,D. castellii)植酸酶的菌株,促进卡氏德巴利酵母植酸酶的研究及工业应用。【方法】将卡氏德巴利酵母植酸酶基因进行密码子优化后转入毕赤酵母GS115中,通过筛选多拷贝、敲除蛋白酶、过表达分子伴侣及转运蛋白的方法获取优势菌株。【结果】所得重组菌株GS115/DCphy(ΔPep4)(BFR2)的产酶酶活是低拷贝菌株的7倍。【结论】研究结果为卡氏德巴利酵母植酸酶的异源表达及潜在工业应用提供了一定的指导。     

植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22．3％,达422IU／ml发酵液的突变菌株黑曲霉N14,其最适pH值为2．5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1．5kb的特异性产物。以pMD18-T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBank Accession：AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5’端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。     

Phytase fromAspergillus niger

132 microorganisms, isolates from soil and decayed fruits, were tested for phytase production. All isolates intensively producing active extracellular phytase were of fungal origin. The most active fungal isolates with phytase activity were identified asAspergillus niger. At the end of the growth phase, the extracellular phytase activity produced byA. niger strain 92 was 132 nkat/mL, with strain 89 it was 53 nkat/mL. In both strains the extracellular enzyme activity exhibited two marked activity optima at pH 1.8 and 5.0 and a temperature optimum at 55°C.     

Characterisation of a thermostable and proteolysis resistant phytase from Penicillium polonicum MF82 associated with the marine sponge Phorbas sp.

Abstract

Phytases are widely used in human and animal nutrition, aquaculture, soil amendment, and in the production of lower myo-inositol phosphates for clinical purposes. Some of these applications, especially feed industry require robust enzymes. Since the marine environments are less studied compared to terrestrial environments, we evaluated the extracellular phytase activity of 110 marine derived filamentous fungal (MDFF) strains previously isolated from sponge and sediment samples of the Turkey. MDFF strains were qualitatively screened for their extracellular phytase activities and P. polonicum MF82 phytase was further characterized following partial purification. Optimum pH and temperature were determined as 5.5 and 60?°C respectively. A significant relative phytase activity was observed in the presence of urea and acetone. However, there was no phytase activity followed by the treatment with Triton X-100 and Tween 80. Characterization studies revealed that P. polonicum MF82 phytase has superior properties for industrial use including wide pH and temperature range for activity, high optimum activity temperature, high thermal and pH stability, resistance to many enzyme inhibitors including various heavy metals, denaturants, detergents, proteases and organic solvents. Phytase extracellularly produced by P. polonicum MF82 strain presents a good candidate for commercial applications. This study demonstrates that the MDFF strains are prolific sources for phytase and presents the first report about the production and characterization of the phytase from a marine-derived P. polonicum strain.     

枯草芽孢杆菌中怀植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化,此中性植酸酶的反应最适pH为7．5,最适温度为55度,在37度下以植酸钠为底物的Km值为0．19mmol/L,植酸酶活性依赖Ca^2 的存在,酶蛋白的分子量大小约为45kD,纯酶蛋白N端序列为Lys-His-Lys-Leu-Ser-Asp-Pro-Tyr-His-Phe-Thr。     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕酵母系统中的高效表达

本文以黑曲霉（Aspergillus niger)NRRL3135菌株植酸酶基因为对象,通过基因人工合成的方法去除了该基因的内含子与信号肽编码序列,换用在毕赤酵母（Pichia pastoris）中使用频率较高的密码子以优化其表达。该人工合成植酸酶基因（PhyA-as)以N端融合的方式正确插入到毕赤酵母表达载体pPICZαA。通过电击将重组表达载体整合人酵母染色体DNA中得到重组转化子。SDS－PAGE结果与表达产物酶这性质研究表明植酸酶得到分泌表达,且与天然产物性质基本一致。筛选得若干株高产基因工程菌,其中SPAN－Ⅲ菌株达到了在摇庆培养条件下,每毫升发酵液产生165000u植酸酶的水平,基本满足工业化生产的要求。