Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.     

Expression and Purification of Functional Human Mu Opioid Receptor from E.coli

N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3–0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K_D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.     

Proof of function of a putative 3-hydroxyacyl-acyl carrier protein dehydratase from higher plants by mass spectrometry of product formation

The predicted mature portion of a putative 3-hydroxyacyl-ACP dehydratase (DH) from Arabidopsis was linked to an N-terminal poly-histidine-tag and the fusion protein expressed in Escherichia coli. Soluble dehydratase was present on induction at 25 °C and pure dehydratase eluted from a nickel-affinity column in 0.2-0.5 M imidazole. High concentrations of imidazole were necessary to retain enzyme solubility. The dehydratase reaction is reversible and 3-hydroxybutyryl- and 2-butenoyl-ACP substrates were prepared from E. coli apo-ACP. Analysis of these suggested contamination of apo-ACP with dehydratase and an additional reverse-phase chromatographic step was required during acyl carrier protein (ACP) preparation. Activity of purified dehydratase was demonstrated by mass spectrometry using 2-butenoyl-ACP, providing the first functional experimental evidence for plant DH gene sequences.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.     

Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27

A psychrothermotolerant alkaline protease isolated from Bacillus pumilus MP27 with a molecular mass ∼53 kDa was isolated from Southern ocean water samples. It was partially purified by single step TPP with purity fold of 16.65. The enzyme was found to be widely stable within a range of temperature and pH, maintaining 52.25% of its activity at 50 °C and 92% at pH 12. The enzyme exhibited an exceptional activity along with tested detergents, showing 98% stability with SDS (10 mg/ml) and ̴ 99% stability with Tide detergent (7 mg/ml). Further, the alkaline protease gene of 1152 bp was successfully cloned in pGEM-T Easy vector in E. coli DH5α. The gene sequence was further translated, modeled and molecular dynamic simulation was performed. The modeled protein was highly unstable during the first 5 ns and therefore could not able to form bonds with the ligand after 1 ns of simulation.     

Expression and single-step purification of mercury transporter (merT) from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l⁻¹.     

High yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli

Heparinase I from Flavobacterium heparinum, a source of diverse polysaccharidases, suffers from low yields, insufficient purity for structural studies and insolubility when expressed as a recombinant product in Escherichia coli that is devoid of glycosaminoglycan polysaccharidases. In this study, cDNA coding for the orthologue of F. heparinum heparinase I was constructed from genomic information from the mammalian gut symbiont Bacteroides thetaiotaomicron and expressed in E. coli as a fusion protein with GST at the N-terminus. This resulted in high yield (30 mg/g dry bacteria) of soluble product and facilitated one-step affinity purification to homogeneity. Purified heparinase I bearing the GST fusion exhibited a K_m of 2.3 μM and V_max of 42.7 μmol/min with a specific activity of 164 U/mg with heparin (average 12,000 Da) as substrate. The results indicate a 2-fold improvement in yield, specific activity and affinity for heparin as substrate over previous reports. The data suggest that the heparinase I from the gut symbiont exhibits a higher intrinsic affinity for heparin than that from F. heparinum. The purified GST fusion enzyme exhibited a requirement for Ca²⁺ and a pH optimum between 6.7 and 7.3 that was similar to the enzyme freed of the N-terminal GST portion. Our study revealed that catalytic activity of heparinase I requires a reducing environment. The GST facilitated immobilization of heparinase I in solid phase either for clinical purposes or for structural studies in absence of interference by contaminating polysaccharidases.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

Enhanced secreting expression and improved properties of a recombinant alkaline endoglucanase cloned in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An alkaline endoglucanase from Bacillus akibai III-3A was successfully expressed in Escherichia coli in active form, and secretion was greatly enhanced by addition of 5 g/l ethylenediamine tetraacetic acid (EDTA) to the culture medium at the induction time of 12 h. Under the optimal culture conditions, extracellular and total endoglucanase activities were 18.5 and 31.2 U/ml, respectively. Both the recombinant and native enzymes exhibited similar properties with respect to broad pH stability, good thermostability, and resistibility to various metal ions and reagents examined. However, unlike the native endoglucanase that was partly inhibited by sodium dodecyl sulfate (SDS), the recombinant enzyme had good resistibility to SDS, being very stable in the commercial detergents, and no decrease in residual activity was observed in 0.2% (w/v) laundry detergent, indicating that it was suitable for application in detergents industry.     

A novel surfactant-, NaCl-, and protease-tolerant β-mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HJ14

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0–30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0–30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5–2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.     

Purification,characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

Background

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).

Methods

The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.

Results

The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The K_m V_max and K_cat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s^− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a K_i as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan^r exhibited almost 50% reduction in intracellular trehalose concentration.

Conclusion

A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.

General significance

This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.     

The effects of intertidal air exposure on the respiratory physiology and the killing activity of hemocytes in the pacific oyster, Crassostrea gigas (Thunberg)

The occurrence of summer mortalities of the commercially important Pacific oyster, Crassostrea gigas, has increased in recent years. These mortality events occur during the late summer when water temperatures are at their highest. Many theories have been proposed concerning the causes including reproductive stress, environmental stress, disease, or synergistic interactions of these factors. C. gigas are grown intertidally and are exposed to the air (emersed) for hours at a time. These organisms can experience extreme changes in temperature during the course of a day. An oyster closed during emersion depletes the oxygen stores to near zero within the shell and builds up CO₂ causing a decrease in tissue pH. The focus of this study is to determine the respiratory (pH, Po₂, Pco₂ and total CO₂) and immune responses of oysters exposed to air at normal seasonal temperatures, and to determine whether these stresses associated with emersion inhibit the immune system of the oyster and contribute to the summer mortalities. The respiratory variables of the hemolymph of oysters submerged at 18 °C (pH = 7.52 ± 0.04 S.E.M., Po₂ = 7.09 ± 0.53 S.E.M. kPa and Pco₂ = 0.20 ± 0.03 S.E.M. kPa) varied significantly from oysters emersed for four hours at 22°C (pH = 7.11 ± 0.03 S.E.M., Po₂ = 3.83 ± 0.15 S.E.M. kPa, Pco₂ = 0.36 ± 0.03 S.E.M. kPa) and those emersed for four hours at 30 °C (pH = 6.84 ± 0.02 S.E.M., Po₂ = 3.10 ± 0.12 S.E.M. kPa, Pco₂ = 1.31 ± 0.06 S.E.M. kPa). The ability of hemocytes to kill the bacterium Vibrio campbellii was assessed using an in vitro assay to generate a killing index. There was no significant difference in the killing index between pH treatment groups (p = 0.856): at pH 7.6 killing index = 50.2% ± 2.33 S.E.M., at pH 6.6 killing index = 52.3% ± 3.67 S.E.M.. Temperature was the only factor to significantly affect the killing indices among temperature and oxygen treatment groups. The killing index was lowest (29.3% ± 3.25 S.E.M.) at 30 °C and 7% oxygen, simulating in vivo oxygen pressure in well-aerated conditions and 30 °C and 3% oxygen, simulating in vivo oxygen pressure in hypoxia (30.5% ± 3.25 S.E.M.), compared with the index in 7% oxygen at low temperature (18 °C) (44.4% ± 4.50 S.E.M.) or compared with low oxygen (3%) at low temperature (18 °C) (39.7% ± 2.51 S.E.M.). The seasonal and diurnal rise in temperature may, therefore, be an important factor contributing to summer mortalities of C. gigas.     

Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM (AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A k_cat value of 72 ± 4 s⁻¹ and a K_M value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with K_d values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

Activation of Archaeoglobus fulgidus Cu-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu⁺ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other P_IB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu⁺-Cys K_D (< 10^− 10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu⁺ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu⁺, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K_m suggesting no changes in the E₁ ↔ E₂ equilibrium. Characterization of Cu⁺ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu⁺ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu⁺-Cys complex.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Understanding the cause of false negative β‐d‐glucuronidase reactions in culture media containing fermentable carbohydrate

Aims:  To explain the basis for false negative β‐glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Methods and Results:  Escherichia coli strains were assessed for their reactions in culture media containing a β‐d ‐glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β‐d ‐glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β‐d ‐glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose‐containing media expressed virtually no β‐d ‐glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. Conclusions:  E. coli that failed to produce green colonies on MLGA produced lower levels of β‐d ‐glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Significance and Impact of the Study:  Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β‐d ‐glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).     

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.