Domains involved in the in vivo function and oligomerization of apical growth determinant DivIVA in Streptomyces coelicolor

The coiled-coil protein DivIVA is a determinant of apical growth and hyphal branching in Streptomyces coelicolor . We have investigated the properties of this protein and the involvement of different domains in its essential function and subcellular targeting. In S. coelicolor cell extracts, DivIVA was present as large oligomeric complexes that were not strongly membrane associated. The purified protein could self-assemble into extensive protein filaments in vitro . Two large and conspicuous segments in the amino acid sequence of streptomycete DivIVAs not present in other homologs, an internal PQG-rich segment and a carboxy-terminal extension, are shown to be dispensable for the essential function in S. coelicolor . Instead, the highly conserved amino-terminal of 22 amino acids was required and affected establishment of new DivIVA foci and hyphal branches, and an essential coiled-coil domain affected oligomerization of the protein.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Electrical excitability of artificial enzyme membranes: II. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes

This paper deals with aspects of the reciprocal interaction between enzyme activity and the microenvironment or the potential difference in artificial proteinaceous membranes bearing cross-linked acetylcholinesterase. The potential difference resulting from asymmetric substrate injection into the system is recorded as a function of time. The influence of the membrane charge density on both enzyme activity and potential difference is studied by varying the external solution pH. The enzyme specific potential is initiated by local change of pH at the membrane level and the dependence on the buffer strength is studied. The recorded potential difference appears to be the result of the reciprocal interaction between enzyme reaction and the diffusion of substrate or products.     

A flow-through enzymatic microreactor immobilizing lipase based on layer-by-layer method for biosynthetic process: Catalyzing the transesterification of soybean oil for fatty acid methyl ester production

A simple and convenient method was proposed in this paper to develop a flow-through enzymatic micro-reactor made from polytetrafluoroethylene (PTFE). It consisted of the polydopamine layer (functioned as a primer) and layer by layer (LBL) coatings composed of polyethylenimine (PEI) and lipase. The multiple deposition of PEI and lipase was the key factor of increasing the enzyme loading on microreactor. After 8 PEI/lipase layers, enzyme loading on the inner surface of 5-m microchannel reached a maximum (350 μg to 400 μg), compared with approximately 20 μg in single layer. Microreactor with higher enzyme loading was successfully applied on transesterification of soybean oil for effective fatty acid methyl ester (FAME, biodiesel) production. A 95.2% conversion rate of biodiesel can be achieved in 53 min under optimized conditions, instead of a couple of hours in the traditional batch reaction.     

Steady-state kinetics of immobilized enzymes at very low substrate concentrations studied using the asarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

A method for determining initial velocities of enzymatic reactions at very low substrate concentrations is presented. It is based on teh continuous perfusiion of substrate-containing media through the enzyme, previously deposited as a thin layer on a solid support. An analytical rationalization of the dependence of the enzymatic activity upon the substrate supply and the flow rate was developed (substrate supply (μmol/min) = flow rate (ml/min) × inflowing substrate concentration (μmol/ml). This paper shows that a straight line should be expected from a double-reciprocal plot of the velocity of the enzymatic reaction and flow rate. The reciprocal of the ordinate at the origin is the strict initial velocity for a given, constant, and very low substrate concentration, since substrate consumption and product accumulation tend to zero. Results obtained with two different sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase preparations agree with the theoretical predictions. The method enabled the use of ATP concentrations in the range of 10^?8 M: it required neither an ATP-regerating system nor the dilution of the enzyme protein, and it presented no limitations for the reaction time. Both ATPase preparations showed two apparent K_m values for the substrate in the submicromolar and micromolar ranges: 0.25–12.0 μM for the purified ATPase, and 0.17–1.65 μM for the microsomal ATPase.     

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40 °C. A maximum GOS of 30% (dry basis) was obtained after 60 h of reaction time, 50 g/L ILC, 241 U of enzyme (specific loading of 600 U/g-membrane), and 0.5 mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20 °C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.     

Physical and chemical immobilization of choline oxidase onto different porous solid supports: Adsorption studies

This work carries out for the first time the comparison between the physical and chemical immobilization of choline oxidase onto aminated silica-based porous supports. The influence on the immobilization efficiency of concentration, pH, temperature and contact time between the support and choline oxidase, was evaluated. The immobilization efficiency was estimated taking into consideration the choline oxidase activity, which was assessed by using cadmium telluride (CdTe) quantum dots (QDs), obtained by hydrothermal synthesis, as photoluminescent probes. Hydrogen peroxide produced by enzyme activity was capable of quenching CdTe QDs photoluminescence. The magnitude of the PL quenching process was directly related with the enzyme activity.By comparing the chemical process with the physical adsorption, it was observed that the latter provided the highest choline oxidase immobilization. The equilibrium data were analyzed using Langmuir and Freundlich isotherms and kinetic data were fitted to the pseudo-first-order and pseudo-second-order models. Thermodynamic parameters, such as Gibbs free energy and entropy were also calculated. These results will certainly contribute to the development of new sensing schemes for choline, taking into account the growing demand for its quantification in biological samples.     

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium

Racemic _DL-tert-leucine (_DL-Tle) was resolved to obtain enantiopure _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, _L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of _L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated _D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.     

Catalytic resolution of (RS)-HMPC acetate by immobilized cells of Acinetobacter sp. CGMCC 0789 in a medium with organic cosolvent

Kinetic resolution of a chiral alcohol, 4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopentenone (HMPC), a key intermediate for the production of prallethrin insecticides, was successfully carried out by enantioselective hydrolysis of (RS)-HMPC acetate using calcium alginate gel-entrapped cells of a newly isolated esterase-producing bacterium Acinetobacter sp. CGMCC 0789. When the effect of different cosolvents was investigated, it was found that isopropanol could markedly enhance the activity and enantioselectivity of the immobilized cells. The optimum concentration of isopropanol was 10% (v/v) where immobilized cells still showed good operational stability. After 10 cycles of reaction, no significant decrease in the enzyme activity was observed. The catalytic specificity constants (V_max/K_m) for both enantiomers of the substrate were determined with partially purified enzyme, giving 0.0184 and 0.671 h⁻¹ for the (S)- and (R)-ester, respectively.