The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Covalent coupling method for lipase immobilization on controlled pore silica in the presence of nonenzymatic proteins

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica previously activated with glutaraldehyde in the presence of nonenzymatic proteins. This strategy is suggested to protect the enzyme from aggregation effects or denaturation that occurs as a result of the presence of silane precursors used in the formation of the silica matrix. The immobilization yield was evaluated as a function of the lipase loading and the additive type (albumin and lecithin) using statistical concepts. In agreement with the mathematical model, the maximum coupling yield (32.2%) can be achieved working at high lipase loading (450 units x g(-1) support) using albumin as an additive. In these conditions, the resulting immobilized lipase exhibits high hydrolytic (153.2 U x mg(-1)) and esterification (337.6 mmol x g(-1) x min) activities. The enhanced activity of the final lipase derivative is the sum of the benefits of the immobilization (that prevents enzyme aggregation) and the lipase coating by additives that increases the accessibility of active sites to the substrate.     

Effects of cationic polymer on performance of UASB reactors treating low strength wastewater

The effect of cationic polymer additives on biomass granulation and COD removal efficiency had been examined in lab-scale upflow anaerobic sludge blanket (UASB) reactors, treating low strength synthetic wastewater (COD 300-630 mg/l). Under identical conditions, two reactors were operated with and without polymer additives in inoculum under four different organic loading rates (OLRs). The optimum polymer dose was adopted based upon the results of jar test and settling test carried out with inoculum seed sludge. With the use of thick inoculum, SS greater than 110 g/l and VSS/SS ratio less than 0.3, granulation was observed in UASB reactor treating synthetic wastewater as well as actual sewage, when OLR was greater than 1.0 kg COD/m(3) d. Polymer additive with such thick inoculum was observed to deteriorate percentage granules and COD removal efficiency compared to inoculum without polymer additives. At OLR less than 1.0 kg COD/m(3) d, proper granulation could not be achieved in both the reactors inoculated with and without polymer additive. Also, under this low loading, drastic reduction in COD removal efficiency was observed with polymer additives in inoculum. Hence, it is rational to conclude that biomass granulation for treatment of low strength biodegradable wastewater depends on the applied loading rate and selection of thick inoculum sludge.     

Effect of the enzyme form on the activity,stability and enantioselectivity of lipoprotein lipase in toluene

Summary The properties of various forms of lipoprotein lipase (powder, adsorbed onto Celite, covalently linked to PEG, with additives) in toluene were investigated. The form of the enzyme dramatically influenced the activity and stability of the enzyme with the highest activity obtained with PEG-lipase and the highest stability with Celite-immobilized lipase. By contrast, the enantioselectivity was only marginally affected.     

Additive Effect of Dextrans on the Stability of Horseradish Peroxidase

The influence of various concentration (10, 20, and 30% w/v) of different molar weighted dextrans as additives on the stability of HRP has been studied in aqueous medium. Native HRP preparations were formulated with different additives for storage stabilization and better performance at high temperature and pH. The results obtained show a stabilizing effect in the presence of an additive (75 kDa dextran). The enzyme with 75 kDa dextran (in concentration 10% w/v) showed the highest thermal resistance and the best performance for long-term storage at pH 5.0. In the presence of the 75 kDa dextran, the enzyme activity was increased threefold at 25 °C and lost only 15% activity in 2 h at 50 °C in comparison to the native enzyme which lost all its activity. In addition, dextran protected HRP against inactivation by air bubbles.     

Intramolecular esterification by lipase powder in microaqueous benzene: factors affecting activity of pure enzyme

Various factors affecting the catalytic activity of pure lipase of Pseudomonas fluorescens in microaqueous benzene were investigated with respect to lactonization of 15-hydroxypentadecanoic acid. Without deposition of the enzyme or of the enzyme plus activity enhancer (additive) on celite powder, the pure enzyme was very poorly dispersed in the microaqueous benzene, resulting in very low activity. The enzyme immobilized on celite powder exhibited the highest activity at a free water content of ca. 0.083%. When a sugar alcohol such as erythritol, arabitol, or sorbitol was added before lyophilization with approximate proportion of 3 g/g enzyme, marked increases in the enzyme activity were observed at a shifted optimal free water content, i.e., 0.04%. Inclusion of phosphotidylcholine resulted in a somewhat higher activity than in the system of enzyme plus celite only. Addition of lactose, bovine serum albumin, casein, dextran, polyvinyl alcohol, phosphate, or NaCl all caused a decrease in the enzyme activity. From the effects of the additives examined, it is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water-immiscible organic solvent: (1) optimum moisture content, (2) disperser (support particles having enough surface area on which the enzyme is thinly deposited), and (3) activity enhancer (additive) at optimum concentration The importance of noting the purity of the enzyme preparation is emphasized when its catalysis in an organic solvent is investigated.     

Characteristics of proteinaceous additives in stabilizing enzymes during freeze-thawing and -drying

Protein-stabilizing characteristics of sixteen proteins during freeze-thawing and freeze-drying were investigated. Five enzymes, each with different instabilities against freezing and dehydration, were employed as the protein to be stabilized. Proteinaceous additives generally resulted in greater enzyme stabilization during freeze-thawing than sugars while the degree of stabilization for basic lysozyme and protamine were inferior to that of neutral and acidic proteins. Freeze-drying-induced inactivation of enzyme was also reduced by the presence of a proteinaceous additive, the extent of which was lower than that for a sugar. In both freeze thawing and freeze drying, the enzymes stabilization by the proteinaceous additive increased with increasing additive concentration. The enhancement of enzyme inactivation caused by pH change was also reduced in the presence of proteinaceous additives. The combined use of a sugar such as sucrose and dextran tended to increase the stabilizing effect of the proteinaceous additive.     

Stabilization of Penicillium occitanis cellulases by spray drying in presence of Maltodextrin

The stabilization of fungal cellulases by spray drying, the thermal stability of Penicillium occitanis cellulases and the effect of some additives were studied. We observed that CMCase activity presents a good stability at 50 degrees C, even after 60 h of incubation. On the other hand, beta-glucosidase activity was more sensitive (loss of 50%) and reacts on total cellulases activities (Filter paper activities). The addition of hydrophilic agents such as ethylene glycol, polyethylene glycol (PEG6000) enhanced enzyme activity. The effect of PEG and Maltodextrin, another water activity decreasing agent, were then tested during the spray drying of Pol6 cellulases. The presence of 1% PEG allowed the best recovery but had a negative effect on enzyme stability while 1% Maltodextrin showed a negative effect on enzyme recovery but a very positive effect on enzyme stabilization.     

Thermodynamic, kinetic, and operational stabilities of yeast alcohol dehydrogenase in sugar and compatible osmolyte solutions

Thermodynamic, kinetic, and operational stabilities of yeast alcohol dehydrogenase (YADH) were measured and compared in aqueous solutions containing various sugars (sucrose, glucose, and ribose) and compatible osmolytes (betaine and sarcosine). In the measurement of operational stability, native YADH was entrapped and physically immobilized in an ultrafiltration hollow fiber tube to retain the native characteristics of the enzyme. All the additives tested increased thermodynamic stability and kinetic stability of YADH. The order of the magnitude of stabilization effect among additives was different between thermodynamic and kinetic stabilities. Compared to the thermodynamic and kinetic stabilities, the effects of additives were much different in operational stability. Sucrose, glucose, and betaine stabilized YADH substantially while ribose and sarcosine destabilized the enzyme. These results show that the thermodynamic and kinetic stabilities do not necessarily guarantee the operational stability of YADH. The coexistence of stabilizing solute was proved effective to increase the productivity of the bioreactor with immobilized YADH.     

Thermodynamic,kinetic, and operational stabilities of yeast alcohol dehydrogenase in sugar and compatible osmolyte solutions

Thermodynamic, kinetic, and operational stabilities of yeast alcohol dehydrogenase (YADH) were measured and compared in aqueous solutions containing various sugars (sucrose, glucose, and ribose) and compatible osmolytes (betaine and sarcosine). In the measurement of operational stability, native YADH was entrapped and physically immobilized in an ultrafiltration hollow fiber tube to retain the native characteristics of the enzyme. All the additives tested increased thermodynamic stability and kinetic stability of YADH. The order of the magnitude of stabilization effect among additives was different between thermodynamic and kinetic stabilities. Compared to the thermodynamic and kinetic stabilities, the effects of additives were much different in operational stability. Sucrose, glucose, and betaine stabilized YADH substantially while ribose and sarcosine destabilized the enzyme. These results show that the thermodynamic and kinetic stabilities do not necessarily guarantee the operational stability of YADH. The coexistence of stabilizing solute was proved effective to increase the productivity of the bioreactor with immobilized YADH.     

Preparation of a robust biocatalyst of d-amino acid oxidase on sepabeads supports using the glutaraldehyde crosslinking method

In this work different protocols to immobilize d-amino acid oxidase (DAAO) on sepabeads were assayed (ionic adsorption on different supports and covalent attachment using glutaraldehyde), studying the stability of the final preparations. The highest stability was achieved by the treatment with glutaraldehyde of DAAO adsorbed on Sepabeads EA (a commercial aminated support having ethylendiamine groups). In fact, this derivative was six times more stable than the enzyme adsorbed only by ionic interaction and much more stable than the soluble enzyme. The effect of the nature of the amino groups in the support was then analyzed. DAAO adsorbed on sepabeads coated with polyethylenimine (PEI) yielded a higher stability than the preparation on Sepabeads EA. The treatment with glutaraldehyde of DAAO adsorbed on Sepabeads PEI yielded the best results in terms of stability, being 200 times more stable than DAAO adsorbed onto Sepabeads EA. The effects of polyethylenimine size and glutaraldehyde concentration were also studied. sepabeads coated with 25 kDa polyethylenimine and treatment with 0.5% glutaraldehyde solution were the optimal parameters regarding the stability (the half life time was 9 h at 56° C, 600 times more stable than the soluble enzyme). Moreover, the optimal derivative showed a maximum load capacity of 15 mg/g of support. This derivative seems to fulfill the requirements for industrial applications.     

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.     

Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin

Glucoamylase (GA) was immobilized by adsorption on carbon support: on Sibunit, on bulk catalytic filamentous carbon (bulk CFC) and on activated carbon (AC). This was used to prepare heterogeneous biocatalysts for the hydrolysis of starch dextrin. The effect of the texture characteristics and chemical properties of the support surface on the enhancement of the thermal stability of the immobilized enzyme was studied, and the rates of the biocatalyst's thermal inactivation at 65-80 degrees C were determined. The thermal stability of glucoamylase immobilized on different carbon supports was found to increase by 2-3 orders of magnitude in comparison with the soluble enzyme, and decrease in the following order: GA on Sibunit>GA on bulk CFC>GA on AC. The presence of the substrate (dextrin) was found to have a significant stabilizing effect. The thermal stability of the immobilized enzyme was found to increase linearly when the concentration of dextrin was increased from 10 wt/vol % to 50 wt/vol %. The total stabilization effect for glucoamylase immobilized on Sibunit in concentrated dextrin solutions was about 10(5) in comparison with the enzyme in a buffer solution. The developed biocatalyst, 'Glucoamylase on Sibunit' was found to have high operational stability during the continuous hydrolysis of 30-35 wt/vol % dextrin at 60 degrees C, its inactivation half-time (t1/2) exceeding 350 h. To improve the starch saccharification productivity, an immersed vortex reactor (IVR) was designed and tested in the heterogeneous process with the biocatalyst 'Glucoamylase on Sibunit'. The dextrin hydrolysis rate, as well as the process productivity in the vortex reactor, was found to increase by a factor of 1.2-1.5 in comparison with the packed-bed reactor.     

Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*

Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint covalent attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20 degrees C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins (all non-covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint covalent attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.     

Optimisation of the composition of a screen-printed acrylate polymer enzyme layer with respect to an improved selectivity and stability of enzyme electrodes

Glucose oxidase (GOD) was immobilized on screen-printed platinum electrodes by entrapment in a screen printable paste polymerized by irradiation with UV-light. The influences of different additives, in particular polymers and graphite, on the sensitivity and stability of the sensor and the permeability of the enzyme layer for a possible electrochemical interferent were investigated. The chosen additives were Gafquat 755N, poly-L-lysine, bovine serum albumin (BSA), sodium dodecylsulfate (SDS), polyethylene glycol (PEG), Nafion and graphite. All additives led to increases of glucose signals, i.e. improved the sensitivity of glucose detection with Gafquat 755N, poly-L-lysine, SDS and graphite showing the strongest influences with increases by a factor 4, 6.5, 5 and 10, respectively. Ascorbic acid was used as a model interferent showing the influence of the enzyme layer composition on the selectivity of the sensor. The addition of Gafquat 755N or poly-L-lysine led to higher signals not only for glucose, but also for ascorbic acid. SDS addition already reduced the influence of ascorbic acid, which was almost completely eliminated when Nafion (5%) and PEG (10%) were added. A comparable beneficial effect on the selectivity of the sensors was also observed for the addition of 0.5% graphite. Thus, the enzyme electrodes with PEG, Nafion or graphite as additives in the enzyme layer were applied to glucose determinations in food samples and samples obtained from E. coli cultivations.     

Modulation of egg-white lysozyme activity by viscosity intensifier additives

The activity of egg-white lysozyme was measured in the presence of carbohydrate additives in the reaction medium. These additives show a significant affinity for water. They depress water activity and increase the viscosity of the medium. Solute-solvent interactions in aqueous solutions of the additives are characterized by properties such as the intrinsic viscosity, Huggins constant apparent molar volume and hydration number. It was found that, despite the lowering of enzyme activity when the concentration of additive is increased, the behavior remains Michaelian and neither modification of Km nor inhibition by excess substrate is observed. On the other hand, the effect of the viscosity of the medium on enzyme activity was determined. This effect is independent of the nature of the additive at high viscosities (greater than 4 mPa s-1) for which enzyme activity is very low and appears to vary according to the kind of additive in dilute solution at low viscosities (less than 2 mPa s-1).     

Urease immobilization on macroporous silicas

Urease was immobilized on macroporous silicas using gamma-aminopropyl triethoxysilane and glutaraldehyde. The amount of protein on the surface, the structure of pores of the support and the purity of the initial enzyme were varied, the enzymic activity of the immobilized preparations being controlled. After the immobilization of sufficiently large quantities of the enzyme (about 3 mg protein per m2 support) about 35% of the specific activity was retained. The maximum activity per unit weight of the support was observed for silicagels and silochromes with the mean diameter of pores 70-90 nm and the specific surface area about 70 m2/g. The use of purified urease produced highly active preparations of the immobilized enzyme (17,000 U per g dry support). Freeze-drying of the immobilized enzyme in the presence of sorbitol yielded dry preparations retaining their activity.     

Freeze denaturation of enzymes and its prevention with additives

Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media.     

Effect of the Support Size on the Properties of β-Galactosidase Immobilized on Chitosan: Advantages and Disadvantages of Macro and Nanoparticles

The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. β-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. β-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.     

Effect of mass-transfer limitations on the selectivity of immobilized alpha-chymotrypsin biocatalysts prepared for use in organic medium

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.