Surface modification of polyacrylonitrile with nitrile hydratase and amidase from Agrobacterium tumefaciens

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; ¹³C labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25°C, 4°C and -20°C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.     

Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli.Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding α-hydroxy amides and acids while retaining enantiopurity.     

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.     

对羟基苯乙腈水合酶高产菌株的选育

从全省各处采集的50多份土样筛选到一株产腈水合酶能力较高的菌株E10a,该菌株产生的腈水合酶为非诱导酶,产酶条件优化实验结果表明,产酶培养基组成:麦芽糖20 g/L,酵母膏5 g/L,尿素7.5 g/L,味精0.75 g/L,K2HPO40.5 g/L,KH2PO40.5 g/L,MgSO40.5 g/L,FeSO4.7H2O 10 mg/L,CoCl210 mg/L,微量元素母液0.8 ml/L;最佳培养条件为:培养温度28℃,摇床转速150 r/min,培养基起始pH 7.0,培养时间5 d,在优化培养条件下,1 h可将1 g/L质量浓度的底物对羟基苯乙腈全部转化为对羟基苯乙酰胺。     

一类生物催化剂——氰基耐受型腈水合酶

氰基耐受型腈水合酶是一类生物催化剂。与普通腈水合酶相比,它能够耐受体系中较高浓度的氰基而不受抑制,从而为α-羟(氨)基酰胺的工业化合成开辟了崭新途径。研究腈水合酶的氰基耐受性机理及提高其耐受能力是目前需要解决的关键问题。综述了腈水合酶受氰基抑制的机制,氰基耐受型腈水合酶的发现以及其在蛋氨酸和2-羟基异丁酰胺生物合成中的应用。同时,对今后氰基耐受型腈水合酶基础、应用研究的思路进行了探讨。     

红球菌低分子量型腈水合酶的异源激活及激活子的结构域功能

腈水合酶激活子具有亚基自身交换伴随子或者金属离子伴随子的功能,能够辅助腈水合酶摄取金属离子,对于腈水合酶的活性表达必不可少。与腈水合酶自身相比,激活子的序列保守性低,研究其激活作用的特点,探索其结构与功能之间的关系,对于理解腈水合酶的成熟机制具有重要意义。将红球菌Rhodococcus rhodochrous J1低分子量型腈水合酶L-NHase分别与4种异源激活子组合共表达,测定异源激活子对L-NHase的激活作用,进一步对激活子进行序列分析和结构模拟,并研究关键结构域的功能。结果表明,4种异源激活子均能激活L-NHase,但激活后L-NHase的比酶活存在差异,激活子A对L-NHase的激活程度最高,激活后的L-NHase比酶活为出发酶的97.79%;激活子G对L-NHase的激活程度最低,激活后的L-NHase比酶活为出发酶的23.94%。激活子E和激活子G具有保守结构域TIGR03889,缺失其中部分序列会使两者的激活作用基本丧失。将激活子G的N端序列替换为激活子E的N端序列,并将激活子E的C端序列添加至激活子G的C端,能够使L-NHase的比酶活提高178.40%。激活子的激...     

Fed-batch production and simple isolation of succinoglycan from Agrobacterium tumefaciens

Succinoglycan production by Agrobacterium tumefaciens was increased from 9.5 g/l to 14 g/l when the microorganism was cultivated under fed-batch condition, as compared to batch fermentation. Cells and broth impurities were removed from the fermentation broth with activated charcoal at a temperature of 90°C, yielding a clear and odorless solution of the polymer. Viscosity mesurement, NMR spectroscopy and Mw determination proved that the polymer retains its main features, practically unaffected by the heat treatment.     

一株腈水合酶产生菌的培养及酶转化试验

诺卡氏菌KY1023能利用乙腈作为生长的碳源和氮源.最适培养条件为(g·L-1)葡萄糖10,醇母膏20,玉米浆10,诱导剂10,MgSO4·7H2O 0.5,K2HPO40.5,KH2PO40.5,pH7.0,菌株在28℃、250rpm条件下培养24h,丙烯酰胺酶活力达到1330μ/ml.休眠细胞在3h以内,可积累丙烯酰胺浓度达到250g·L-1,乙酰胺浓度达到400g·L-1.     

抗生素对菊花叶片再生及农杆菌生长的影响

以3个菊花品种的试管苗叶片为材料,通过探讨5种抗生素硫酸卡那霉素,氨苄青霉素,头孢噻肟钠,头孢唑林钠和羧苄青霉素对其再生的影响,结果表明,硫酸卡那霉素浓度为5mg/L时能完全抑制JB、Jc的再生,15mg/L时能完全抑制JA的再生。其它4种抗生素对3个菊花品种再生影响差异很大。随着浓度升高,头孢噻肟钠使JA、JB再生率持续下降,再生率持续上升;头孢唑林钠使JA、JB、Jc再生率都持续下降;羧苄青霉素使JA、JB、Jc再生率先上升,后下降,均在400mg/L时达到峰值。对于农杆菌LBA4404,头孢噻肟钠的抑菌效果最好,其次是羧苄青霉素。     

Transformation of rice mediated by Agrobacterium tumefaciens

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing competent cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

Rhodococcus rhodochrous J1腈水合酶在大肠杆菌中的表达策略

针对红球菌低分子量腈水合酶（L—NHase）在重组茵中难以表达这一问题,通过对其d亚基及调控蛋白NhlE基因的核糖体结合位点和0c,口亚基间隔序列的长度进行改造,构建了重组表达载体,实现了L．NHase及其调控蛋白NhlE在E．coliB121（DE3）中过量表达。通过培养条件优化,得到最佳表达条件为：37℃培养茵体浓度（DD600）到1．0时,加入终浓度为0．1g／L的CoCl2·6H,0,0．6mmol／L的IPTG,然后在24℃下诱导表达24h。最终得到的重组蛋白粗酶液的活性为（109．9-I-5．5）U／rag。采用Strep．tag／Strep—Tactin亲和层析简化了L-NHase的纯化方法,本研究结果为一些难于异源重组表达的多亚基蛋白质的表达具有一定的借鉴意义。     

根癌农杆菌介导Bt基因转化水稻的研究

为了培育出无筛选标记基因的转基因水稻,试验将loxp-hpt-loxp基因与成基因连锁在-起转化水稻方法,得到loxp-hpt—loxp—Bt转基因水稻植株,再与同质的带有ere基因的水稻杂交,以定向删除潮霉素抗性筛选标记。试验表明以水稻品种“皖粳97”为供试材料,将成熟胚来源的愈伤组织用根癌农杆菌EHA105／pCAMBIA1305．1感染后,筛选出抗性愈伤组织并获得再生植株。经PCR验证,得到20棵转基因水稻植株。     

The plant cell defense and Agrobacterium tumefaciens

We previously identified changes in gene expression in Ageratum conyzoides plant cells inoculated with Agrobacterium tumefaciens by using cDNA-AFLP. Here, we show that a subset of defense-related genes is differentially regulated by an Agrobacterium attachment-deficient mutant. The expression pattern triggered by this mutant is similar to that induced by inoculation with non-pathogenic bacteria. We also observed that the expression level of the defense genes was inversely correlated with the efficiency of transformation by Agrobacterium. We propose that the plant defense system has an important role in controlling infection and transformation and that Agrobacterium may dampen some plant defense responses.     

Media development for large scale Agrobacterium tumefaciens culture

A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH₄)₂SO₄), magnesium sulfate heptahydrate (MgSO₄*7H₂O), calcium chloride dihydrate (CaCl₂*2H₂O), iron (II) sulfate heptahydrate (FeSO₄*7H₂O), manganese (II) sulfate monohydrate (MnSO₄*H₂O), zinc sulfate heptahydrate (ZnSO₄*7H₂O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na₂HPO₄/KH₂PO₄). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 ± 0.01 h^?1 and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract‐peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that l ‐serine and l ‐asparagine were depleted from the media first, followed by l ‐alanine and l ‐glutamic acid. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1218–1225, 2017     

Ti plasmid type affects T-DNA processing in Agrobacterium tumefaciens

Agrobacterium tumefaciens can transfer the T-DNA region of a Ti plasmid to a recipient plant cell. An accepted model that describes the T-DNA transfer mechanism proposes that single-stranded T-complexes are transferred to a recipient plant via a conjugation-like mechanism. This model has been based on examination of a limited number of Ti plasmids. In this study, the type of processed T-DNA molecule created from multiple Ti plasmids was determined. The form of the processed T-DNA was found to vary and was correlated with whether the T-DNA region was organized as a single continuous region or two adjacent regions.     

影响根癌农杆菌转化的因素及其在单子叶作物上的应用

在植物转基因方法中,根癌农杆菌介导的遗传转化应用最为广泛,进一步提高其转化频率并扩大其宿主范围到禾谷类作物是人们所关注的问题。有多种因素影响根癌农杆菌的转化频率,包括植物的受伤反应、细菌的吸附、致病基因的诱导、植物细胞ＤＮＡ合成及修复的活力、外植体的状态等。最近的研究结果证明在适宜的条件下,根癌农杆菌还是可以有效地转化禾谷类作物。本文试就这两方面的研究进展作一论述。     

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens

AIMS: To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. METHODS AND RESULTS: Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. CONCLUSIONS: In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.     

农杆菌vir基因诱导因子研究进展

在众多遗传转化法中,农杆菌(Agrobacterium tumefaciens)介导法以易操作、低费用、插入片段明确、拷贝数低等独特优点成为植物遗传转化的首选。然而,至今仍有许多物种不能被农杆菌转化。研究表明,农杆菌的转化能力是由位于染色体基因组之外Ti质粒上的vir基因决定的。在所有vir基因中,除virA和virG组成型表达外,其它vir基因的表达均需酚类化合物的诱导;糖类物质可增强酚类化合物对vir基因的诱导;低磷酸和酸性pH环境也可促进vir基因的诱导表达。文章论述了酚类化合物、糖类物质、低磷酸、酸性pH和培养温度等因素对农杆菌vir基因诱导表达的影响,以期为更好地利用这一天然载体及为提高转化效率提供依据。     

Tumor Transformation of Petunia hybrida via Pollen Co-Cultured with Agrobacterium tumefaciens

Pollen preparations usually show a high nuclease activity. Therefore, to avoid DNA degradation, co-cultures of pollen and Agrobacterium tumefaciens were evaluated as a new tool in gene transfer experiments. As a model system, Petunia pollen was co-cultured with an A. tumefaciens wild strain. The co-cultured pollen was used for pollination of Petunia flowers. The seeds obtained were germinated and one cotyledon per seedling was removed and put stalk upwards on nutrient agar. In 80% of the cotyledons a callus developed from the cut surface of the stalks which was screened for tumor transformation on hormone-free medium. In repeated subculturing some calli maintained growth on hormone-free medium. Two of these calli were habituated. One callus, the best growing one, showed on Southern blot analysis a distinct hybridization signal at 3.2 kb when probed with Hind III fragment 22 DNA, covering two genes responsible for hormone free growth. This is the exact size that could be expected when plant material has been transformed with T-DNA. Another callus gave a hybridization signal at 2 kb which could only be explained with chromosomal rearrangement. In these two calli there was no co-transformation of the nos-gene: nopaline synthase activity could be detected from none of the calli, and none of the calli DNAs hybridized when probed with the nos-gene. Bacterial contamination could be excluded by probing the DNAs with virfragments.