Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp

An internal loop airlift reactor (ILALR) is developed and studied for biodegradation of phenol/m-cresol as single and dual substrate systems under batch and fed batch operation using an indigenous mixed microbial strain, predominantly Pseudomonas sp. The results showed that the culture could degrade phenol/m-cresol completely at a maximum concentration of 600mgl(-1) and 400mgl(-1), respectively. Batch ILALR study has revealed that phenol has been preferentially degraded by the microbial culture rather than m-cresol probably owing to the toxic effect of the later. Sum kinetic model evaluated the interaction between the phenol/m-cresol in dual substrate system, which resulted in a high coefficient of determination (R(2)) value >0.98). The fed batch results showed that the strain was able to degrade phenol/m-cresol with maximum individual concentrations 600mgl(-1) each in 26h and 37h, respectively. Moreover for fed batch operation, degradation rates increased with increase in feed concentration without any lag in the degradation profile.     

Unstable steady state operations of substrate inhibited cultures by dissolved oxygen control

Microorganism kinetic growth characterized by substrate inhibition was investigated by means of a continuous stirred tank reactor equipped with a feedback controller of the medium feeding flow rate. The aerobic growth of Pseudomonas sp. OX1 with phenol as carbon/energy source was adopted as a case study to test a new control strategy using dissolved oxygen concentration as a state variable. The controller was successful in steadily operating bioconversion under intrinsically unstable conditions. A simple model of the controlled system was proposed to set the feedback controller. The specific growth rate of Pseudomonas sp. OX1 was successfully described by means of the Haldane model. The regression of the experimental data yielded μ(M)=0.26 h(-1), K(Ph)=5×10(-3)g/L and K(I)=0.2g/L. The biomass-to-substrate fractional yield as a function of the specific growth rate did not change moving from substrate-inhibited to substrate-deficient state. The data was modelled according to the Pirt model: m=1.7×10(-2)g/(gh), Y(X/Ph)(Th)=1.3g/g. The specific growth rates calculated for batch and continuous growth were compared.     

Copper removal by algae Gelidium, agar extraction algal waste and granulated algal waste: kinetics and equilibrium

Biosorption of copper ions by an industrial algal waste, from agar extraction industry has been studied in a batch system. This biosorbent was compared with the algae Gelidium itself, which is the raw material for agar extraction, and the industrial waste immobilized with polyacrylonitrile (composite material). The effects of contact time, pH, ionic strength (IS) and temperature on the biosorption process have been studied. Equilibrium data follow both Langmuir and Langmuir-Freundlich models. The parameters of Langmuir equilibrium model were: q(max)=33.0mgg(-1), K(L)=0.015mgl(-1); q(max)=16.7mgg(-1), K(L)=0.028mgl(-1) and q(max)=10.3mgg(-1), K(L)=0.160mgl(-1) respectively for Gelidium, algal waste and composite material at pH=5.3, T=20 degrees C and IS=0.001M. Increasing the pH, the number of deprotonated active sites increases and so the uptake capacity of copper ions. In the case of high ionic strengths, the contribution of the electrostatic component to the overall binding decreases, and so the uptake capacity. The temperature has little influence on the uptake capacity principally for low equilibrium copper concentrations. Changes in standard enthalpy, Gibbs energy and entropy during biosorption were determined. Kinetic data at different solution pH (3, 4 and 5.3) were fitted to pseudo-first-order and pseudo-second-order models. The adsorptive behaviour of biosorbent particles was modelled using a batch reactor mass transfer kinetic model, which successfully predicts Cu(II) concentration profiles.     

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.     

Enzymatic saccharification of solid residue of olive mill in a batch reactor

This paper describes the enzymatic hydrolysis of solid residue of olive mill (OMRS) in a batch reactor with the Trichoderma reesei enzyme. Before enzymatic saccharification, crude lignocellulosic material is submitted to alkaline pre-treatment with NaOH. Optimum conditions of the pre-treatment (temperature of T=100 degrees C and OMRS-NaOH concentration ratio of about R=20) were determined. The optimum enzymatic conditions determined were as follows: pH of about 5, temperature of T=50 degrees C and enzyme to mass substrate mass ratio E/S=0.1g enzyme (g OMRS)(-1). The maximum saccharification yield obtained at optimum experimental conditions was about 50%. The experimental results agree with Lineweaver Burk's formula for low substrate concentrations. At substrate concentrations greater than 40gdm(-3), inhibitory effects were encountered. The kinetic constants obtained for the batch reactor were K(m)=0.1gdm(-3)min(-1) and V(m)=800gdm(-3).     

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 μM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 μM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 μM. Lactate and ethanol were also accumulated at 760 μM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 μM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 μM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 μM h(-1) and K(s)=5.8 and 13.1 μM for control and bioaugmented sludges, respectively).     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and 4-chlorophenol (4-cp) as a cometabolised compound by Pseudomonas putida ATCC 49451 was accomplished by augmenting the medium with conventional carbon sources such as sodium glutamate and glucose. Compared with phenol as the sole carbon source, the addition of 1 gl(-1) sodium glutamate increased the toxicity tolerance of cells toward 4-cp and significantly improved the biodegradation rates of both phenol and 4-cp even when the initial concentration of 4-cp was as high as 200 mgl(-1). On the other hand, supplementation of glucose caused a significant drop in the medium pH from 7.2 to 4.3 resulting in a reduction of degradation rate, leaving a considerable amount of 4-cp undegraded when the initial concentration of 4-cp was higher than 100 mgl(-1). By regulating the pH of the medium, however, enhancement of degradation rates of phenol and 4-cp in the presence of glucose was achieved with a concomitant complete degradation of phenol and 4-cp.     

Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65 degrees C

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 degrees C. A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1). At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1). The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1). Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)). Additionally, yield coefficients for oxygen and ammonium are reported.     

Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani

Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (K(m)=33.24 μm, k(cat)=1.3 s(-1), K(i)=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting K(m) values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.     

Isolation and selection of phenol-degrading microorganisms from industrial wastewaters and kinetics of the biodegradation

Among 22 species of microorganisms isolated from phenol-containing wastewaters, Candida parapsilopsis was found to be capable of growth on a medium with 1 g/L phenol. Kinetic parameters of phenol biodegradation in a batch reactor were determined by measuring biomass growth rates and phenol concentration as a function of fermentation time. The Haldane equation described cell growth adequately, with kinetic constants mumax = 0.174/h, KS = 11.2 mg/L and Ki = 298 mg/L.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.     

Mutation of Candida tropicalis by irradiation with a He-Ne laser to increase its ability to degrade phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 x 10(7) cells ml(-1) were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter(-1) phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter(-1). The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Biodegradation of phenol in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1 immobilized in an air-stirred reactor with clarifier

Phenol biodegradation by suspended and immobilized cells of Rhodococcus erythropolis UPV-1 was studied in discontinuous and continuous mode under optimum culture conditions. Phenol-acclimated cells were adsorbed on diatomaceous earth, where they grew actively forming a biofilm of short filaments. Immobilization protected cells against phenol and resulted in a remarkable enhancement of their respiratory activity and a shorter lag phase preceding active phenol degradation. Under optimum operation conditions in a laboratory-scale air-stirred reactor, the immobilized cells were able to completely degrade phenol in synthetic wastewater at a volumetric productivity of 11.5 kg phenol m(-3) day(-1). Phenol biodegradation was also tested in two different industrial wastewaters (WW1 and WW2) obtained from local resin manufacturing companies, which contained both phenols and formaldehyde. In this case, after wastewater conditioning (i.e., dilution, pH, nitrogen and phosphorous sources and micronutrient amendments) the immobilized cells were able to completely remove the formaldehyde present in both waters. Moreover, they biodegraded phenols completely at a rate of 0.5 kg phenol m(-3) day(-1) in the case of WW1 and partially (but at concentrations lower than 50 mg l(-1)) at 0.1 and 1.0 kg phenol m(-3) day(-1) in the cases of WW2 and WW1, respectively.     

Immobilized white-rot fungal biodegradation of phenol and chlorinated phenol in trickling packed-bed reactors by employing sequencing batch operation

The biodegradation of phenol and 2,4,6-trichlorophenol (2,4,6-TCP) by immobilized white-rot fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and 2,4,6-TCP (800 and 85 mg l(-1)) concentrations were removed by greater than 98% in 24-30 h batch cycles. Comparable performance between the packing materials was shown. Increased lignin peroxidase (LiP) activity was detected with the introduction of the compounds and optimum activity corresponded to optimum removal periods. Higher LiP activity (16.7-19 Ul(-1)) was detected in glass bead-packed reactor compared to wood chip reactor (0.2-5 Ul(-1)). The presence of Mn(2+) in the wood material possibly effected elevated manganese peroxidase (MnP) activity (0.3-5.8 Ul(-1)) compared to low to negligible activity in the glass bead reactor. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to induce and sustain fungal enzyme activity in inert vs. organic material packed systems.     

Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150

The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled. Both of these compounds can serve as the sole source of carbon and energy for this microorganism. The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol. Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate. Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene. Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150. Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.     

Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor

A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing. Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments. Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts. Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation. Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day. The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates. Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested. In this case, after wastewater conditioning (i.e. pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day.     

Laboratory evaluation of a two-stage treatment system for TCE cometabolism by a methane-oxidizing mixed culture

The objective of this research was to evaluate several factors affecting the performance of a two-stage treatment system employing methane-oxidizing bacteria for trichloroethylene (TCE) biodegradation. The system consists of a completely mixed growth reactor and a plug-flow transformation reactor in which the TCE is cometabolized. Laboratory studies were conducted with continuous growth reactors and batch experiments simulating transformation reactor conditions. Performance was characterized in terms of TCE transformation capacity (T(C), g TCE/g cells), transformation yield (T(Y), g TCE/g CH(4)), and the rate coefficient ratio k(TCE)/K(S,TCE) (L/mg-d). The growth reactor variables studied were solids retention time (SRT) and nutrient nitrogen (N) concentration. Formate and methane were evaluated as potential transformation reactor amendments. Comparison of cultures from 2- and 8-day SRT (nitrogen-limited) growth reactors indicated that there was no significant effect of growth reactor SRT or nitrogen availability on T(C) or T(Y), but N-limited conditions yielded higher k(TCE)/K(S,TCE). The TCE cometabolic activity of the 8-day SRT, N-limited growth reactor culture varied significantly during a 7-year period of operation. The T(C) and T(Y) of the resting cells increased gradually to levels a factor of 2 higher than the initial values. The reasons for this increase are unknown. Formate addition to the transformation reactor gave higher T(C) and T(Y) for 2-day SRT growth reactor conditions and significantly lower T(C), T(Y), and k(TCE)/K(S,TCE) for 8-day SRT N-limited conditions. Methane addition to the transformation reactor inhibited TCE cometabolism at low TCE concentrations and enhanced TCE cometabolism at high TCE concentrations, indicating that the TCE cometabolism in the presence of methane does not follow simple competitive inhibition kinetics. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 650-659, 1997.     

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

Treatment of phenolic wastewater in UASB reactor: effect of nitrogen and phosphorous

Phenolic wastewater representing an industrial wastewater was supplemented with varying amount of nitrogen (N) and phosphorous (P) and treated in upflow anaerobic sludge blanket reactor (UASBR). The variation of COD:N:P from 300:10:1 to 300:1:0.1, did not influence the conversion of phenol COD to methane COD. The concentration of N and P in the influent was reduced from 25.5mgl(-1) to 2.5mgl(-1) and 2.5 to 0.25mgl(-1), respectively. However, on further reducing the nutrients in the feed from 300:1:0.1 to 300:0:0 the (i) CH(4)-COD decreased from 90% to 40%, and (ii) cell yield reduced to 25-50%. The average cell yield was 3.5%. Percent N and P in cells varied from 10% to 14% and 0.6% to 2.4%, respectively. The activity of the sludge assessed as specific methanogenic activity (SMA) was found in the range from 0.15 to 0.66g CH(4)-CODg(-1) VSSd(-1). The optimum COD:N:P for phenolic wastewater has been estimated to be 300:1:0.1.     

Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors

Phenylalanine ammonia-lyase (PAL) catalyzes the beta-elimination of ammonia from L-phenylalanine to trans-cinnamic acid. A study of inhibition of PAL by phenol, ortho-cresol, and meta-cresol gave mixed inhibition; para-cresol is not an inhibitor. The calculated values of K(i) and alphaK(i) are phenol, K(i)=2.1+/-0.5 mM and alphaK(i)=3.45+/-0.95 mM; ortho-cresol, K(i)=0.8+/-0.2 mM and alphaK(i)=3.4+/-0.2 mM; meta-cresol, K(i)=2.85+/-0.15 mM and alphaK(i)=18.5+/-1.5 mM. The synergistic inhibition of the same inhibitors with glycine showed a lack of inhibition with the para-cresol/glycine pair, while mixed inhibition was observed with the ortho-cresol/glycine pair (K(i)=0.038+/-0.008 mM, alphaK(i)=0.13+/-0.04 mM) and phenol/glycine pair (K(i)=0.014+/-0.003 mM, alphaK(i)=0.058+/-0.01 M). The meta-cresol/glycine pair gave competitive inhibition (K(i)=0.36+/-0.076 mM). The strong synergistic inhibition observed implies that the inhibitors bind at the active site: in fact, the inhibitors used imitate the structure of the substrate. The order of synergistic inhibition is the same for the sites related to K(i) and alphaK(i). These results are in agreement with the inhibitors entering two active sites located in two different subunits.