A Novel and Simple Method for the Purification of Extracellular Levansucrase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

A new and simple method for the purification of extracellular levansucrase from Zymomonas mobilis from highly viscous fermentation broth was developed. After incubation of the fermentation broth with a fructose-polymer cleaving enzyme preparation (Fructozyme, Novozymes, DK) for 48 h, levansucrase precipitated as aggregates and was redissolved in a 3 M urea solution. By ongoing size-exclusion chromatography on Sephacryl S-300 the final levansucrase preparation was purified 100-fold and exhibited a specific activity of 25–35 U/mg_protein. The levansucrase was stable in 3 M urea solution for at least four months without inactivation. To maximize the enzyme yield the dynamic changes of extracellular levansucrase activity during fermentation were investigated. The highest levansucrase activity was observed during the logarithmic phase of growth (15–19 h of fermentation).Received: 26 September 2002 / Accepted: 24 October 2002     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Congo Red attached monosize poly(HEMA-co-MMA) microspheres for use in reversible enzyme immobilisation

Monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) (poly(HEMA-co-MMA)), microspheres were prepared by dispersion polymerisation of HEMA and MMA in an ethanol–water medium in the presence of an initiator (α,α′-azobisisobutyronitrile, AIBN). An affinity dye, i.e. Congo Red (CR) was attached covalently and then Fe³⁺ ions were incorporated. The poly(HEMA-co-MMA)-CR attached and poly(HEMA-co-MMA)-CR-Fe³⁺ incorporated microspheres were used in the immobilisation of glucose oxidase (GOD) via adsorption. The adsorption capacities of these microspheres were determined by varying the concentration of GOD in the adsorption medium. GOD adsorption capacities of the Fe³⁺ incorporated microspheres (165 mg g⁻¹) was greater than that of the dye-attached microspheres (126 mg g⁻¹). The non-specific adsorption of the GOD on the poly(HEMA-co-MMA) microspheres was negligible. The K_m values for both immobilised poly(HEMA-co-MMA)-CR-GOD (7.2) and poly(HEMA-co-MMA)-CR-Fe³⁺-GOD (6.8) were higher than that of the free enzyme (6.6 mM). Optimum reaction pH was 5.0 for free and 7.0 for both immobilised preparations. Optimum reaction temperature of the adsorbed enzymes was 10 °C higher than that of the free enzyme and was significantly broader. After 10 successive uses the retained activity of the adsorbed enzyme was 93%. It was observed that enzyme could be repeatedly adsorbed and desorbed on the CR attached poly(HEMA-co-MMA) microspheres without significant loss in adsorption capacity or enzyme activity.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.     

Chemical Modification Causes Similar Change in Dependence on Water Activity of Chymotrypsin Hydration and Catalysis in Hexane

Amino groups in alpha-chymotrypsin were reacted with pyromellitic anhydride, introducing 17 to 32 additional carboxyl groups. This modification causes a major change in the water adsorption isotherm of the lyophilized protein powder. Little water is bound by the modified enzyme at water activity (a_w) below 0.35, but it shows increased water binding at a_w over 0.5. This correlates with a similar change in the a_w dependence of the catalytic activity of the enzyme powder suspended in hexane, with a much steeper increase in activity of the modified chymotrypsin.     

A new alcohol dehydrogenase with unique stereospecificity from Pseudomonas sp.

A new nicotinamide cofactor-dependent alcohol dehydrogenase from Pseudomonas strain SBD6 (PADH) was isolated and purified 150-fold to homogeneity using a combination of salt precipitation, anion-exchange chromatography, gel filtration chromatography, and dye matrix chromatography. Approximately 10 mg of pure enzyme can be obtained from 10 g of wet cells. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, and potassium cyanide result in complete loss of activity. The enzyme is very stable (t_1/2 7 days at pH 7 and 25°C in the absence of 2-propanol and 18 days in the presence of 10% 2-propanol, v/v) and possesses a broad substrate specificity with transfer of the pro-(R) hydride from NADH to the si face of carbonyl substrates to give (R)-alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Synthetic scale reductions are facilitated with 2-propanol as a hydride source for the regeneration of NADH. The kinetic mechanism is ordered bi-bi with the cofactor binding first. Based on NAD and 2-propanol, the kinetic parameters of the enzyme were determined to be V_max = 29.9 Units mg⁻¹ at 25°C and pH 8.5, K_m^NAD = 0.36 m and K_m^2-propanol = 0.19 m .     

Immobilisation of lipases by adsorption and deposition: high protein loading gives lower water activity optimum

Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (mol min^–1 mg protein) when Celite was used as support and 2.3 (mol min^–1 mg^–1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40–100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g^–1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 mol min^–1 mg^–1 protein) at a _w=0.11, while an enzyme preparation with low protein loading (4 mg g^–1) showed highest specific activity at a _w=0.75.     

Chemical modification of alpha-chymotrypsin for obtaining high transesterification activity in low water organic media

Alpha-chymotrypsin was made more hydrophilic by modifying 11 (out of 16) ε-amino groups with pyromellitic dianhydride. The hydrophilic preparation was precipitated with n-propanol. This preparation gave significantly higher initial rates at the optimum a_w (127.51 nmol mg^?1 min^?1 in n-octane and 21.30 nmol mg^?1 min^?1 in acetonitrile at a_w=0.33) compared with the lyophilized preparation (53.50 nmol mg^?1 min^?1 in n-octane and 0.26 nmol mg^?1 min^?1 in acetonitrile at a_w=0.97). FT-IR showed that the precipitate of modified alpha-chymotrypsin has a higher content of alpha-helices and beta-sheets compared to the lyophilized powder.     

Kinetic Modeling of Acetophenone Reduction Catalyzed by Alcohol Dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp.

NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (k_d=0.0141 min⁻¹) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.     

Adsorptive control of water in esterification with immobilized enzymes: II. fixed-bed reactor behavior

Experimental and theoretical studies are conducted to understand the dynamic behavior of a continuous-flow fixed-bed reactor in which an esterification is catalyzed by an immobilized enzyme in an organic solvent medium. The experimental system consists of a commercial immobilized lipase preparation known as Lipozyme as the biocatalyst, with propionic acid and isoamyl alcohol (dissolved in hexane) as the reaction substrates. A complex dynamic behavior is observed experimentally as a result of the simultaneous occurrence of reaction and adsorption phenomena. Both propionic acid and water are adsorbed by the biocatalyst resulting in lower reaction rates. In addition, an excessive accumulation of water in the reactor leads to a rapid irreversible inactivation of the enzyme. A model based on previously-obtained adsorption isotherms and kinetic expressions, as well as on adsorption rate measurements obtained in this work, is used to predict the concentration and thermodynamic activity of water along the reactor length. The model successfully predicts the dynamic behavior of the reactor and shows that a maximum thermodynamic activity of water occurs at a point at some distance from the reactor entrance. A cation exchange resin in sodium form, packed in the reactor as a selective water adsorbent together with the catalyst particles, is shown to be an effective means for preventing an excessive accumulation of water formed in the reaction. Its use results in longer cycle times and greater productivity. As predicted by the model, the experimental results show that the water adsorbed on the catalyst and on the ion exchange resin can be removed with isoamyl alcohol with no apparent loss in enzyme activity.     

Fast and efficient adsorption/desorption of protein by a novel magnetic nano-adsorbent

A novel magnetic nano-adsorbent was prepared by covalently binding polyacrylic acid (PAA) on Fe₃O₄ superparamagnetic nanoparticles (13.2 nm) via carbodiimide activation. The maximum weight ratio of PAA to Fe₃O₄ was 0.12 (i.e., average of two PAA molecules on a magnetic nanoparticle). The magnetic nano-adsorbent possessed a high ionic exchange capacity of 1.64 meq g^–1 and was efficient for the recovery of lysozyme. The lysozyme could be completely adsorbed in 0.1 M phosphate buffer at pH 3–5 and completely desorbed in NaSCN solution (>1 M) within 1 min, and retained 95% activity after adsorption/desorption. In addition, the adsorption behavior followed the Langmuir adsorption isotherm with a maximum adsorption amount of 0.224 mg mg^–1 and a Langmuir adsorption equilibrium constant of 10 ml mg^–1 at 25 °C. The change of enthalpy at 15–35 °C was –4.2 kJ ml mol^–1 mg^–1.     

Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.     

Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir

Summary A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R(+)-phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg²⁺ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on NADPH and was inactive when supplied with NADH as the coenzyme. Important enzymological data of the dehydrogenase are: K _m (acetophenone) 0.6 mM, K _m (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of NADPH: glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.     

Effect of amino acid substitutions at the active center on the activity of Bacillus stearothermophilus alcohol dehydrogenase

Catalysis of the thermostable alcohol dehydrogenase from Bacillus stearothermophilus is performed by a proton release system involving a zinc-bound water molecule, a hydroxyl group of Thr40 (threonine position at 40), and an imidazole ring of His43. Amino acid residues (Thr40 and His43) at the active center were substituted by Ser and Arg, respectively. Thr40Ser had a tendency toward lower activity for primary alcohols than the wild type enzyme. However, the mutant enzyme became more active for substrates with a larger side chain, such as 2-methyl-1-propanol and cyclohexanol. This phenomena might be explained by the fact that the methyl group of Thr40 was eliminated in Ser. His43Arg exhibited higher activity to primary alcohols (except 2-methyl-1-propanol) and acetaldehyde (as a reverse reaction) than the wild type, but little activity for secondary alcohols and ketones. The K_m value for ethanol (K_m-e) of His43Arg was fifty-fold larger than that of the wild type. The characteristics of these mutant enzymes are also discussed.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Lipase hydration state in the gas phase: sorption isotherm measurements and inverse gas chromatography

The adsorption of water and substrate on immobilized Candida antarctica lipase B was studied by performing adsorption isotherm measurements and using inverse gas chromatography (IGC). Water adsorption isotherm of the immobilized enzyme showed singular profile absorption incompatible with the Brunauer-Emmet-Teller model, probably due to the hydrophobic nature of the support, leading to very low interactions with water. IGC allowed determining the evolution with water thermodynamic activity (a(W)) of both dispersive surface energies and acidity and basicity constants of immobilized enzyme. These results showed that water molecules progressively covered immobilized enzyme, when increasing a(W), leading to a saturation of polar groups above a(W) 0.1 and full coverage of the surface above a(W) 0.25. IGC also enabled relevant experiments to investigate the behavior of substrates under a(W) that they will experience, in a competitive situation with water. Results indicated that substrates had to displace water molecules in order to adsorb on the enzyme from a(W) values ranging from 0.1 to 0.2, depending on the substrate. As the conditions used for these adsorption studies resemble the ones of the continuous enzymatic solid/gas reactor, in which activity and selectivity of the lipase were extensively studied, it was possible to link adsorption results with particular effects of water on enzyme properties.     

Sterol carrier protein hypothesis: requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis.

The 105,000 x g supernatant (S₁₀₅) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S₁₀₅ and required for this conversion. We have now resolved this controversy by the discovery that S₁₀₅ contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein₁ (SCP₁), sterol carrier protein₂ (SCP₂), and sterol carrier protein₃ (SCP₃). SCP₁ is required for the microsomal conversion of squalene to lanosterol, SCP₂ for the microsomal conversion of 4,4-dimethyl-Δ⁸-cholesterol to C₂₇-sterols, and SCP₃ for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.     

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 g_{Laminaribiose} g_sugar^?1 in the desorbate with a total productivity of 5.6 mg_{Laminaribiose} L_{enzyme bed}^?1 h^?1 without the use of recycles.     

Variation in amount of enzyme protein in natural populations

Among strains of Drosophila melanogaster each derived from a single fertilized female taken from natural populations, there is variation in both alcohol dehydrogenase (ADH) activity and the amount of ADH protein. The correlation between ADH activity and number of molecules over all strains examined is 0.87 or 0.96 in late third instar larvae depending on whether the substrate is 2-propanol or ethanol. With respect to the two common electrophoretic allozymic forms, F and S, segregating in these populations, the FF strains on the whole have higher ADH activities and numbers of ADH molecules than the SS strains. Over all strains examined, enzyme extracts from FF strains have a mean catalytic efficiency per enzyme molecule higher than that of enzyme extracts from SS strains when ethanol is the substrate, and much higher when 2-propanol is the substrate. One FF strain had an ADH activity/ADH protein ratio characteristic of SS strains.     

Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.