L-异亮氨酸和甘氨酸对大肠杆菌表达与分泌邻苯二酚2,3-双加氧酶的作用

证实了甘氨酸与L-异亮氨酸对大肠杆菌表达邻苯二酚2,3-双加氧酶(CatO_2ase)的促进作用和甘氨酸促使该酶分泌至胞外培养基中的作用.产酶量高低和分泌量多少与培养基种类、甘氨酸和L-异亮氨酸的浓度以及培养时间等因素有关.在甘氨酸存在的情况下,胞壁对溶菌酶的敏感性有所增加,超微形态似有变化,还存在其他物质的伴随分泌,故甘氨酸可能是引起细胞壁结构的改变而导致邻苯二酚2,3-双加氧酶等胞内容物被动分泌至胞外.     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

邻苯二酚2,3-双加氧酶的结构和功能研究进展

邻苯二酚是所有芳香族化合物降解过程中的重要的中间产物,其降解有邻位和间位裂解两条裂解途径,分别由邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)催化裂解。本综述简要介绍了邻苯二酚2,3-双加氧酶的结构和功能的研究进展。     

产胞外邻苯二酚1，2-双加氧酶的菌种筛选和发酵条件的研究

从112株细菌中筛选出两株产胞外邻苯二酚1,2一双加氧酶的假单胞菌(Pseubomonassp.)。 进行了该菌产酶的发酵条件试验。产酶的最适温度为30℃,最适起始pH为6．8—7-0。葡萄糖、麦芽糖和甘油对产酶有明显的抑制作用,苯甲酸钠对产酶有促进作用。氨态氮对菌体生长和产酶是必需的。琥珀酸钠是酶形成的有教诱导物。采用0.15％苯甲酸钠培养基(pH6.8—7.0),于30℃振荡培养72h,每毫升发酵液酶活力可达10单位。     

邻苯二酚1,2-双加氧酶的结构、功能及同工酶现象研究进展

邻苯二酚是芳香族化合物多条生物降解途径中共有的一种重要的中间产物,根据开环方式的不同,可分为邻位降解途径和间位降解途径,其中邻位降解途径中的关键酶是邻苯二酚1,2-双加氧酶。本文主要综述了邻苯二酚1,2-双加氧酶的结构、酶学性质,以及它在芳香烃降解菌中存在的同工酶现象及其功能研究进展。     

邻苯二酚2,3—双加氧酶在大肠杆菌的表达与定域

本文在大肠杆菌/枮草芽孢杆菌间的穿梭质粒pTG 402的基础上构建了几个新的带有显色标志基闲xylE的表达质粒,摸索了该基因所编码的邻苯二酚2,3-双加氧酶(CatO_2ase)的表达条件,分析了该酶一级结构与二级结构的亲水性和疏水性,测定了它在大肠杆菌中的产量与分布。结果表明,CatO_2ase与各质粒的表达量不等,表达量高低与培养时间、宿主菌及诱导与否等影响因素有关;表达后有部分酶可在胞外测出,但大部分仍定域于膜内,亲、疏水性分析示该酶不具分泌性蛋白的显著特点。因该酶易于检测和定量,可作为一种选择性标记和监测指示系统在基因工程中推广应用,同时亦为用基因工程菌消除芳烃类化合物的污染提供了理论依据。     

食酸丛毛单胞菌AN3菌株降解苯胺代谢途径的研究

食酸丛毛单胞菌(\%Comamonas acidovorans\%)AN3菌株中降解苯胺的酶类均为诱导酶,在以苯胺为唯一碳、氮源和能源生长的细胞中,含有苯胺双加氧酶、邻苯二酚2,3双加氧酶、2羟基己二烯半醛酸脱氢酶、4草酰巴豆酸脱羧酶和4羟基2酮戊酸醛缩酶等。苯胺双加氧酶作用于苯胺的\%K\%m值和\%V\%\-\{max\}分别为292μmol/L和3.57μmol\5mg\+\{-1\}·min-1;邻苯二酚2,3双加氧酶作用于邻苯二酚的\%K\%m和\%V\%\-\{max\}分别为16.4 mol/L和15.2μmol\5mg\+\{-1\}\5min\+\{-1\}。根据实验结果,推测了该菌株降解苯胺的代谢途径。     

固定化胞外邻苯二酚1，2－双加氧酶的研究

首次将胞外邻苯二酚1,2－双加氧酶固定化,并用于制备顺,顺—己二烯二酸.该固定化酶表观活力高,使用范围扩大,耐酸性及耐碱性都有显著提高,并且使用稳定性好,得到的产物浓度及纯度均较高,酶与产物容易分离,整个工艺简单、独特、新颖,有利于工业化应用.     

微生物降解邻苯二酚的动力学研究

以一株产邻苯二酚双加氧酶的假单胞菌84103为试验菌,对其降解邻苯二酚的动力学进行了研究。结果表明,该降解过程为酶催化反应,测定了反应最适温度、最适pH、饱和菌量、Km值及诱导作用等。     

复合与合成培养基对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

为实现来源于Paenibacillus macerans JFB05-01的α-环糊精葡萄糖基转移酶（α-CGT酶）的高效胞外表达,以含分泌型信号肽OmpA的大肠杆菌E.coli BL21（DE3）{pET-20b（+）/α-cgt}为研究对象,比较了其在不同诱导条件下复合与合成培养基中生长产酶的规律。结果表明在添加甘氨酸的条件下采用合成培养基,以0.8 g/L/h的乳糖进行流加诱导所得的胞外酶活和生产强度最高。在该条件下发酵30小时后胞外α-CGT酶的环化活性达113.0U/ml（水解活性为79 100.0IU/ml）,是复合培养基胞外产酶的2.3倍,完全满足工业化生产的需求。     

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.     

Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies.

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.     

Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic Acid

A methanogenic consortium able to use 3-chlorobenzoic acid as its sole energy and carbon source was enriched from anaerobic sewage sludge. Seven bacteria were isolated from the consortium in mono- or coculture. They included: one dechlorinating bacterium (strain DCB-1), one benzoate-oxidizing bacterium (strain BZ-2), two butyrate-oxidizing bacteria (strains SF-1 and NSF-2), two H(2)-consuming methanogens (Methanospirillum hungatei PM-1 and Methanobacterium sp. strain PM-2), and a sulfate-reducing bacterium (Desulfovibrio sp. strain PS-1). The dechlorinating bacterium (DCB-1) was a gram-negative, obligate anaerobe with a unique "collar" surrounding the cell. A medium containing rumen fluid supported minimal growth; pyruvate was the only substrate found to increase growth. The bacterium had a generation time of 4 to 5 days. 3-Chlorobenzoate was dechlorinated stoichiometrically to benzoate, which accumulated in the medium; the rate of dechlorination was ca. 0.1 pmol bacterium day. The benzoate-oxidizing bacterium (BZ-2) was a gram-negative, obligate anaerobe and could only be grown as a syntroph. Benzoate was the only substrate observed to support growth, and, when grown in coculture with M. hungatei, it was fermented to acetate and CH(4). One butyrate-oxidizing bacterium (NSF-2) was a gram-negative, non-sporeforming, obligate anaerobe; the other (SF-1) was a gram-positive, sporeforming, obligate anaerobe. Both could only be grown as syntrophs. The substrates observed to support growth of both bacteria were butyrate, 2-dl-methylbutyrate, valerate, and caproate; isobutyrate supported growth of only the sporeforming bacterium (SF-1). Fermentation products were acetate and CH(4) (from butyrate, isobutyrate, or caproate) or acetate, propionate, and CH(4) (from 2-dl-methylbutyrate or valerate) when grown in coculture with M. hungatei. A mutualism among at least the dechlorinating, benzoate-oxidizing, and methane-forming members was apparently required for utilization of the 3-chlorobenzoate substrate.     

Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Expression of a cell surface antigen from Rhizobium leguminosarum 3841 is regulated by oxygen and pH.

Rhizobium leguminosarum bv. viciae 3841 was grown in liquid suspension culture to investigate how culture conditions could affect the expression of a developmentally regulated cell surface antigen associated with lipopolysaccharide. The antigen, which is recognized by monoclonal antibody AFRC MAC 203, was expressed when cultures were grown at neutral pH under low-oxygen conditions (less than 7.5% [vol/vol] O2 in the gas phase). Antigen was also expressed in aerobically grown cultures at pH values below 5.3. The nature of the nitrogen and the carbon sources had no effect on antigen expression except by indirect changes on the pH of the culture medium; similarly, growth in 0.3 M NaCl did not result in antigen expression. The induction of MAC 203 antigen by low-oxygen or low-pH culture conditions is discussed in the context of tissue-specific expression within the legume root nodule.     

Extracellular factors affecting the adhesion of Pseudomonas fluorescens cells to glass surface

Two factors affecting the adhesion of Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, anti-adhesion (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.     

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.     

Extracellular factors affecting the adhesion ofPseudomonas fluorescens cells to glass surfaces

Two factors affecting the adhesionof Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, antiadhesin (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.