Disulfide bond formation and activation of Escherichia coli β-galactosidase under oxidizing conditions

Escherichia coli β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of Escherichia coli β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.     

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale. Received: 1 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Effect of Glutaraldehyde on Cell Viability, Triphenyltetrazolium Reduction, Oxygen Uptake, and β-Galactosidase Activity in Escherichia coli

Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.     

Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in a Lac⁻mutant of Escherichia coli HB101. Five clones expressing β-glucosidase activity were shown by restriction enzyme analysis to contain a common 4.4 kbp fragment of inserted DNA. Hybridization of recombinant plasmids with chromosomal DNA ratified the physical maps of the inserted DNA and was further used to confirm that the 4.4 kbp fragment was common to all five clones. Enzyme activity, comprising cellobiase and aryl-β-glucosidase, was similar with respect to substrate specificity for each of the five clones, and was expressed independently of the orientation of the cloned DNA. A differential effect of temperature on activity of the cellobiase and aryl-β-glucosidase activities was observed but in other respects, the properties of the cloned β-glucosidase corresponded to those of the single β-glucosidase previously described for C. thermocellum.     

Influence of amylose,amylopectin and pullulan on β-glucuronidase activity of starch-degrading Escherichia coli

Synthesis of -glucuronidase in starch-degrading Escherichia coli (S1) was induced by amylose, amylopectin and pullulan supplied in mineral medium as the sole carbon source (1%, w/v). The maximum activity occurred after 4 days when cultures reached the stationary phase of growth, but induction was also evident during log-phase. The effects obtained with amylose, amylopectin and pullulan were higher than that obtained with maize starch.     

Immobilization of Escherichia coli containing ω-transaminase activity in LentiKats®

Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized with different concentrations of cetrimonium bromide (CTAB) and ethanol; the best results were obtained with CTAB 0.1% which resulted in an increase in reaction rate by 40% compared to the whole cells. The synthesis of PEA was carried out using isopropyl amine (IPA) and L ‐alanine (Ala) as amino donors. Using whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole and permeabilized cells containing ω‐TA and their immobilized LentiKats® counterparts showed similar initial reactions rates and yields in the reaction systems, indicating 100% of immobilization efficiency (observed activity/activity immobilized) and absence of diffusional limitations (due to the immobilization). Immobilization of whole and permeabilized cells containing ω‐TA in LentiKats® allowed improved stability as the biocatalyst was shown to be efficiently reused for five reaction cycles, retaining around 80% of original activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Where do nutrients in an inlet-less lake come from? The water and nutrient balance of a small mesotrophic lake

In this article we verified the hypothesis that precipitation is the main nutrient source in an inlet-less lake. We tested this hypothesis by calculating the water and nutrient (phosphorus and nitrogen) balances of a lake located in a hypsographically diverse moraine landscape (northern Poland). All components of the water and nutrient budgets were measured independently, including precipitation and ground water fluxes. The investigations showed that although precipitation constituted about a half or more of the annual water balance in this inlet-less lake, the ground water inflow and outflow play the most important role in the balance of nutrients. Therefore, critical nutrient loads calculated according to the methodology developed within the OECD Eutrophication Programme, which was focused mostly on drainage-type lakes, appeared inadequate in the case of this small seepage lake. Moreover, studies showed that throughout the investigations, a continuous ground water flow-through occurred in the lake. It questions the possibility of calculating the ground water flow simply as a difference between surface inflows and outflows.     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Phylogenetic grouping and virulence potential of extended-spectrum-β-lactamase-producing Escherichia coli strains in cattle

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.     

Long-term trends in water temperature and ice cover in the subalpine lake, Øvre Heimdalsvatn,and nearby lakes and rivers

Long-term data series of ice cover on lakes and river temperatures from the mountain areas of Norway are lacking. The present study analyses the last four decades of ice data from the subalpine lake, Øvre Heimdalsvatn, and water temperature data from its outlet river, Hinøgla. These data are compared to water temperature data from three neighbouring, quite different locations, the glacier-fed rivers Leirungsåi and Sjoa, and the alpine lake, Bessvatn. The study also examines the air temperature/river temperature relationships, and the air temperature/ice freeze-up and break-up dates. During the months of July, August and September, the water temperature in Hinøgla was well correlated to the air temperature, but the correlation was poor in the remaining months due to the ice cover and snow conditions. A significant temperature increase of 2–3°C has been observed in Hinøgla in the months August–October since 1984. There were only minor changes in the duration of the ice cover season during the last 40 years, but a delay of 9 days was found in the freeze-up date and a delay of 6 days in the break-up date, although the latter was not significant.     

A Monte-Carlo step-by-step simulation code of the non-homogeneous chemistry of the radiolysis of water and aqueous solutions—Part II: calculation of radiolytic yields under different conditions of LET,pH, and temperature

The importance of the radiolysis of water in the initial events following irradiation of biological systems has motivated considerable theoretical and experimental work in the field of radiation chemistry of water and aqueous systems. These studies include Monte-Carlo simulations of the radiation track structure and of the non-homogeneous chemical stage, which have been successfully used to calculate the yields of radiolytic species (H^·, ^·OH, H₂, H₂O₂, e_aq⁻, …). Most techniques used for the simulation of the non-homogeneous chemical stage such as the independent reaction time (IRT) technique and diffusion kinetics methods do not calculate the time evolution of the positions of the radiolytic species. This is a major limitation to their extension to the simulation of the irradiation of radiobiological systems. Step-by-step (SBS) simulation programs provide such information, but they are very demanding in term of computer power and storage capacity. Recent improvements in computer performance now allow the regular use of the SBS method in radiation chemistry simulations. In the first of a series of two papers, the SBS method has been reviewed in details and the implementation of a SBS code has been discussed. In this second paper, the results of several studies are presented: (1) the time evolution of the radiolytic yields from the formation of the radiation track to 10⁻⁶ s; (2) the effect of pH on yields (pH ~ 0.4–7.0); (3) the effect of proton energy (and LET) on yields (300 MeV-0.1 MeV), and iv) the effect of the ion type (¹H⁺, ⁴He²⁺, ¹²C⁶⁺) on yields. Nonbiological applications, i.e., the study of the temperature on the yields (about 25–300°C) and the simulation of the time evolution of G(Fe³⁺) in the Fricke dosimeter are also discussed.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

Combinatorial expression of different β-carotene hydroxylases and ketolases in Escherichia coli for increased astaxanthin production

Journal of Industrial Microbiology & Biotechnology - In natural produced bacteria, β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) convert β-carotene into...