Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

Kinetic behavior of immobilized Penicillin acylase

Penicillin acylase has been immobilized to carboxymethylcellulose and to the resin Amberlite XAD7. The reaction kinetics of the enzyme were affected by both intrinsic (molecular) and microenvironmental effects. The Michaelis constant for the enzyme increased after immobilization as a result of an intrinsic effect of the reagent, glutaraldehyde, used for enzyme immobilization. Microenvironmental effects were of two types: diffusional limitation of access of substrate and a reaction-generated pH depression in the support particles. This depression of internal pH was observed in all the preparations and could be reduced by addition of pH buffering salts to reactor. An adsorbed pH-indicating dyc was used to determine the surface and internal pH of particles of XAD7–penicillin acylase under various reaction conditions. The extent of diffusional rate limitation in XAD7–penicillin acylase was related to the penetration depth of protein into the porous support particles. The penetration depth of protein and thus the diffusional limitation of the reaction rate could be controlled by the conditions of preparation of the immobilized enzyme. A staining technique was used to observe the location of the protein.     

Nisin production by a mixed-culture system consisting of Lactococcus lactis and Kluyveromyces marxianus.

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.     

Profile control scheme in a Bakers' yeast fed-batch culture

This article develops and discusses a practical and useful computer control scheme so that the biomass concentration or the specific growth rate will as accurately as possible follow a desired profile specified in advance. Many computer simulations certified the validity of the proposed control scheme. The control scheme proposed, called "programmed-controller/feedback-compensator (PF) system," consists of a programmed controller that will follow the desired profile unless there is noise or disturbance and a feedback compensator that will compensate the noise and correct error in the model parameters. As the feedback compensator, the model reference adaptive control (MRAC) algorithm was also proposed. The PF system with MRAC, named PF-MRAC, could be used sufficiently for the profile control of the specific growth rate. For the profile control of the cell concentration, "predictive control algorithm" should be added to the PF system, and the consequent control scheme was named as the PFP system. Many numerical examples showed that the PFP system with MRAC, named PFP-MRAC, proposed here worked sufficiently well.     

Enzymatic breakdown of threonine by threonine aldolase

1. The enzyme which splits threonine to acetaldehyde and glycine has been partially purified from rat liver (five- to sixfold purification) and the name threonine aldolase proposed for it. 2. The general properties of threonine aldolase have been studied. The enzyme is unstable to a pH below 5. The pH optimum of the enzyme reaction is at 7.5-7.7. The initial rate of production of acetaldehyde is proportional to the enzyme concentration, and when the enzyme concentration is constant, the production of acetaldehyde is proportional to the time, provided that the substrate is in excess. The enzyme is inhibited by the carbonyl group reagent, hydroxylamine. Attempts to demonstrate that pyridoxal phosphate is a cofactor were unsuccessful. 3. The enzyme splits only L-allothreonine and L-threonine and is inactive against the D-forms of these amino acids. 4. The enzyme reaction on DL-allothreonine follows first order kinetics. From the first order velocity constants and the initial rates of the rates of the reaction at various substrate concentrations the Michaelis constant, Ks, for this substrate has been evaluated. Michaelis constants have also been determined for threonine. 5. The optimum temperature for the enzymatic breakdown of DL-allothreonine at pH 7.65 was found to be 50 degrees C. in phosphate buffer and 48 degrees C. in tris-maleate buffer. The rate of thermal inactivation of the enzyme threonine aldolase obeys a first order reaction. The heat of thermal inactivation was calculated by the aid of the van't Hoff-Arrhenius equation to be 43,000 cal. per mole for the temperature range 41.2-46.6 degrees C. 6. Equivalent amounts of acetaldehyde and glycine were formed from DL-allothreonine and the enzymatic breakdown of DL-allothreonine was found to be irreversible.     

Kinetic characteristics of biochemical cyclic reaction systems: Amplification of substrate cycle system

The amplification of a substrate cycle system with reversible closed reaction of two substrates was represented by mathematical equations. The results are summarized as follows: the amplification was affected especially by the affinity of enzyme and substrate, by the rate constant in rate-limiting reaction step, and by the saturation degree of enzyme by substrate. These amplifications were not simply determined by the values of K(m) and V(max), because each rate parameter in the system can affect the degree of amplification independently. The conclusion is that the "apparent" equilibrium constant of this system cannot be uniquely estimated from only data of K(m) and V(max) even if the reaction occurs in a closed system.     

The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide.

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.     

Lipase catalysed hydrolysis of ricebran oil by free and immobilised enzyme system in batch stirred reactor

A change of the reaction rate was observed for the lipasecatalysed hydrolysis of ricebran oil in a batch stirred tank reactor using immobilized lipase enzyme as compared to free enzyme. The reactor rate was observed to be controlled mainly by factors like temperature, pH, initial enzyme concentration, initial substrate concentration and initial products concentration.     

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.     

The oscillating peroxidase-oxidase reaction in an open system. Analysis of the reaction mechanism.

1. The oscillations in the peroxidase-oxidase reaction in an open system with NADH as the hydrogen donor are caused by the reaction starting and stopping at critical concentrations of the substrates O2 and NADH. The existence of such critical concentrations is typical of branched chain reactions. 2. The critical concentrations of O2 and NADH that determine the initiation of the reaction are mutually dependent. 3. The branching reactions that determine these critical concentrations involve compounds I and II. 4. Superoxide may be involved in the branching reactions by reacting with NADH and ferriperoxidase. At pH 5.1 the rate constant for the latter reaction is determined as 1.5 . 10(5) M-1 . s-1, whereas for the former reaction only an upper limit for the rate constant of 3.5 . 10(4) M-1 . s-1 could be estimated. These relatively low rate constants suggest that alternative branching reactions may also be involved.     

Effect of changes in the pH and carbon dioxide evolution rate on the measured respiratory quotient of fermentations

The total concentration of dissolved carbon dioxide in fermentation broths is one to two orders of magnitude greater than that of oxygen for pH > 6.5. The rate of change in this total concentration can be sufficiently large to produce a discrepancy between the carbon dioxide transfer rate (CTR) across the gas-liquid interface, available from gas analyses, and the carbon dioxide evolution rate (CER) of biomass in the fermentor. The CER is the variable of most interest to fermentation technologists but cannot be measured directly. The CTR is commonly used to yield the measured respiratory quotient (called here the TQ, or transfer quotient). Evaluation of the real underlying respiratory quotient (RQ), however, requiures the unmeasureable CER. Equations defining the problem are presented and are found to accurately predict the discrepancy between the TQ and the RQ in fed-batch fermentations of Escherichia coli. During the exponential growth phase, the TQ is less than the RQ. A changing pH can cause the TQ to be bigger or smaller than the RQ, while pH fluctuations associated with on-off pH controller action make the CTR and hence the TQ noisy. The RQ is estimated on-line during an E. coli fermentation and is shown to be constant during the fermentation, even though the TQ varies greatly. (c) 1992 John Wiley & Sons, Inc.     

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.     

Membrane surface potential and the reactivity of the System II primary electron acceptor to charged electron carriers in the medium

A hypothesis is proposed to explain the change in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase.Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149–166).Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.     

Purification and characterization of a novel proteinase, chymopapain S

Chymopapain (EC 3.4.22.6) possesses an essential and a non-essential thiol group, but enzyme preparations produced hitherto always contained less than two thiol groups. Starting from commercial chymopapain or from papaya latex, we have prepared pure enzyme having two thiol groups, which is demanded by mechanistic investigations. The purification was performed by covalent chromatography on activated thiol-Sepharose column, which specifically binds thiol-containing proteins. Elution was effcted with cysteine in a stepwise manner, first at pH 5 then at pH 8. The pure enzyme had a molecular mass of24 700 as estimated by sodium dodecyl sulfate polyacrylamide gel-electrophoresis. Titration of the pure enzyme with 2,2'-dipyridyl disulfide at pH 9 exhibited a biphasic curve. This indicated that under the conditions employed the nonessential thiol group is more reactive than the essential one, which is a characteristic feature of chymopapain B. On the other hand, the magnitude of the rate constant of the same reaction at pH 4, as well as its pH-dependence, was characteristic of chymopapain A. The enzyme with the hybrid kinetic properties was denoted as chymopapain S. We conclude from the above findings that various forms of chymopapain can be classified by their reaction with 2,2'-dipyridyl disulfide.     

Levansucrase of Bacillus subtilis. Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartly residue as the binding site of the fructosyl group.

A covalently linked fructosyl-enzyme complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH. The fructosyl-enzyme bond was shown to be stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate. This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity. However stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values above 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase. Furthermore, nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl-enzyme bond. Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex. Peptic digestion followed by pronase digestion released a fructosyl-aspartate compound that we have isolated in a high state of purity. The lability of the fructosyl-aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the beta-carboxyl of the aspartate residue. Treatment of the trapped complex with cyanogen bromide released only one fructosylated peptide. The apparent molecular weight of this peptide was estimated to be lower than 10000.     

Enzymatic production of L-serine with a feedback control system for formaldehyde addition

Serine hydroxymethyltransferase (SHMT) in the form of crude extract from a recombinant strain of Klebsiella aerogenes was used for the production of L-serine from glycine and formaldehyde (HCHO). A stirred tank bio-reactor with a continuous feed of HCHO (37%) was employed. Since the performance of the serine bioreactor was heavily dependent on how HCHO was fed, an automatic feedback control system was developed for HCHO delivery utilizing the phenomenon of formol titration. This control procedure was based on the following circumstance: as a bioconversion proceeded, if the rate of HCHO feed was balanced by the rate of serine synthesis so that HCHO concentration was maintained near zero, then there was no pH change in the bioreactor. Once the rate of HCHO addition exceeded that of serine synthesis, the HCHO concentration built up and the excess HCHO reacted with the amino group of an amino acid (e.g. glycine or serine) to produce a Schiff base and a proton which lowered the pH. A pH controller detected and relayed this pH change to the on-off switch of the HCHO feed pump. Thus, HCHO infusion stopped when the pH was lower than the set point, which was the initial pH of the reaction. With this control system, the maximum concentration of HCHO that was reached in the bioreactor was only 1mM-3.3mM depending on the pH and amino acid composition in the bioreactor. Moreover, a decrease in pH also signaled the use of a slower feed rate at which HCHO was to be, delivered once the pH resumed its initial value after excess HCHO was consumed by the reaction. Employing this control system, we have optimized the performance of the serine bioreactor to give a serine titer of 450 g/L with an 88% molar conversion of glycine at a volumetric serine productivity of 8.9 g/L/h.     

Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. pH dependence of NADPH binding and isotope rate effect for beta-ketoacyl reductase

The stopped flow method has been used to determine the pH dependence of the kinetics of the binding of NADPH to chicken liver fatty acid synthase over the pH range 6.0-8.5. The kinetics is consistent with a one-step binding mechanism, and the pH dependence of the second order rate constant indicates that an ionizable group either on the enzyme or on NADPH with a pK alpha of 6.1 is of importance in the binding process. The isotope rate effects have been determined for the steady state reaction with (S)- and (R)-[4-2H] NADPH as substrates and are very small. The pH dependence of the rate constant characterizing the reduction of acetoacetyl by NADPH on the enzyme (beta-ketoacyl reductase) and the isotope rate effects on this constant with (S)-[4-2H]NADPH as substrate also have been measured with the stopped flow method. A small pH-dependent isotope rate effect is found; these results suggest hydride transfer is not rate limiting for the beta-ketoacyl reductase reaction on the enzyme surface. The pH dependence of this rate constant is bell shaped and is very similar to that of the turnover number for the overall reaction; this suggests that the beta-ketoacyl reductase reaction may be partially rate limiting for the overall reaction when the enzyme is saturated with substrates.