Structure and activity of alpha-chymotrypsin and trypsin in aqueous organic media

The effects of different concentrations (20-95%) of organic solvents (ethanol, 1,4-dioxane and acetonitrile) were studied on alpha-chymotrypsin and trypsin from bovine pancreas. The changes in secondary structure were followed by CD measurements, and the apparent Michaelis constants (KMapp) and the stabilities of the enzymes were determined. Significant alterations in the CD spectra were found for both enzymes at the different organic solvent concentrations. The apparent KM values of trypsin and alpha-chymotrypsin decreased as the low solvent concentrations were elevated, but then increased in the presence of higher organic solvent concentrations. The stabilities of the enzymes changed on increase of the organic solvent concentration; trypsin exhibited a higher stability than that of alpha-chymotrypsin in all organic solvents. These results show that at an organic solvent content of 95% the manifestation of an enzyme activity similar to that measured in water can be attributed to the similar compositions of the secondary structural elements.     

Effects of Ca2+ on catalytic activity and conformation of trypsin and alpha-chymotrypsin in aqueous ethanol

The effects of calcium ions on the conformation and catalytic activity of trypsin and alpha-chymotrypsin were studied in aqueous ethanol. The activity of alpha-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of alpha-chymotrypsin did not decrease in the presence of 1.2M CaCl2 and 0.6M CaCl2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

Stability of the insoluble form of uridine kinase coupled to zn2+ or pb2+ ions.

Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein-protein interactions in water-immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe-NH(2) was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol-modified trypsin in organic media containing 1-2% water (v/v). The organic solvent-soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate-organic solvent interface. Peptide mapping of bovine insulin digest by reversed-phase high-performance liquid chromatography definitely showed that enzyme-catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents

Dehydrated enzyme powders have been used extensively as suspensions in organic solvents to catalyze synthetic reactions. Prolonged enzyme activity is necessary to make such applications commercially successful. However, it has recently become evident that the stability and thus activity of many enzymes is compromised in organic solvents. Herein we explore the stability of various hydrolases (i.e., lipases from Mucor meihei and Candida rugosa, -chymotrypsin, subtilisin Carlsberg, and pig-liver esterase) and various formulations (lyophilized powder, cross-linked enzyme crystals, poly(ethylene glycol)-enzyme conjugates) in different organic solvents. The results show a roughly exponential activity decrease for all enzymes and formulations studied after exposure to organic solvents. Inactivation was observed independent of the enzyme, formulation details, and the solvent. In addition, no relationship was found between the magnitude of inactivation and the value of initial activity. Thus, quite active formulations lost their activity as quickly as less active formulations. The estimated half-times (t_1/2) for all enzymes and preparations ranged from 1.8 h for subtilisin C. co-lyophilized with methyl-β-cyclodextrin to 61.6 h for the most stable poly(ethylene glycol)--chymotrypsin preparation. The data here presented indicates that the inactivation is likely not related to changes in enzyme structure and dynamics.     

Enhancement of the organic solvent‐stability of the LST‐03 lipase by directed evolution

LST‐03 lipase from an organic solvent‐tolerant Pseudomonas aeruginosa LST‐03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent‐stability of LST‐03 lipase was attempted by directed evolution. The structural gene of the LST‐03 lipase was amplified by the error prone‐PCR method. Organic solvent‐stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri‐n‐butyrin and which overlaid a plate medium. And the organic solvent‐stability was also confirmed by measuring the half‐life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent‐stability in the presence of DMSO. The organic solvent‐stabilities of mutated LST‐03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half‐lives of the LST‐03‐R65 lipase in the presence of cyclohexane and n‐decane were about 9 to 11‐fold longer than those of the wild‐type lipase, respectively. Some substituted amino acid residues of mutated LST‐03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Procedure for obtaining and catalytic properties of trypsin immobilized in cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized in a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In an organic solvent environment, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-argininine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.     

Advantages of the pre-immobilization of enzymes on porous supports for their entrapment in sol-gels

In this work, we have compared the entrapment of free or previously immobilized glucose oxidase using a sol-gel technique. The preimmobilization was carried out on Sepabeads (a porous support) derivatized with glutaraldehyde as the functional group. The prior immobilization of the enzyme permitted to maintain the enzyme activity intact after the formation of the sol-gel. In fact, only 10% of the enzyme activity was lost whereas the soluble enzyme lost 60% of its initial activity. Additionally, enzyme leakage from the sol-gel matrix was avoided, which was relatively high when entrapping the soluble enzyme (39% of the enzyme activity was released after 16 h of incubation in a buffered solution). Moreover, the immobilized enzyme, inside the porous support, cannot be in contact with the sol-gel, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the Sepabeads support.     

Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

CONVERSION OF TRYPSIN TO A COPPER ENZYME: TYROSINASE/CATECHOL OXIDASE BY CHEMICAL MODIFICATION

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu⁺²) introduced p-aminobenzamidine (pABA- Cu⁺²: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu⁺² preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K_cat, K_m, K_cat/K_m), optimum pH and temperature of the trypsin-pABA-Cu⁺² complex was also investigated.     

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.     

Ionic liquids as alternative co-solvents for laccase: study of enzyme activity and stability

The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.     

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.     

Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization of soluble porcine ALT by chemical modification with mono- and bis-imidates. The apparent transition temperatures (‘T_m’, the temperature where 50% of initial activity was lost in 10 min) for native and DMS-modified ALT were 46 and 56 °C respectively. The effects of water-miscible organic solvents (methanol, dimethylformamide, dimethylsulphoxide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in ALT catalytic activity was observed on reaching a certain threshold concentration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V_max and K_m for 2-oxoglutarate as substrate, determined in 10% (v/v) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 °C with no organic solvent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% (v/v) solvent at 35 °C, the DMS-modified ALT's half-life was up to 4.6 times greater than native enzyme's. DMS-modified ALT was also more stable in urea and guanidine HCl, and its refolding was more noticeable, than that of native enzyme.     

Estimation of protein digestibility—III. Studies on the digestive enzymes from the pyloric ceca of rainbow trout and salmon

Digestive proteinases were isolated and partially purified from the pyloric ceca of trout and salmon. Their stability and some catalytic properties were compared with those of a three-enzyme system that is used for determination of in vitro protein digestibility. In contrast to the three-enzyme system, pyloric ceca trypsin and total proteinase activity were least stable at pH values below 5.0 and most stable under alkaline conditions up to pH 10.0. Thermal inactivation (50%) occurred in 60 min at 55°C for trypsin activity of trout and salmon ceca proteinases and at 40°C for the three-enzyme system at the pH (8.0) of the in vitro assay. Thermal inactivation (50%) of total proteinase activity occurred in 60 min at about 55, 50 and 35°C for chinook, trout and three-enzyme preparations, respectively. SDS-PAGE zymograms of the ceca enzymes showed the presence of several proteolytic activity bands. Two of the bands corresponded in molecular weight to trypsin and chymotrypsin. Ceca proteinases differ from the three-enzyme system in their response to inhibitors; in particular, the ceca proteinases are much more sensitive to soybean trypsin inhibitor than the procine trypsin used in the three-enzyme system when assayed for trypsin, but less sensitive when assayed for total proteinase. The distinctive properties of ceca enzymes help explain why they are more appropriate than the three-enzyme system, and other enzyme cocktails for in vitro protein digestibility assay of saunonid feed components.