Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media

Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc.     

Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules

Summary Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules have been developed for the re-use of an oil-soluble enzyme in organic media. The esterification rate of the surfactant-coated lipase immobilized in the microcapsules was thirty times that of the powder lipase. More than 90% of the enzymatic activity of the capsulated lipases has been maintained after recycling six times.     

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes in supercritical carbon dioxide

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes was performed in supercritical CO(2) and liquid CO(2) at 308.2 and 333.2 K at pressures of 6.1 and 10.1 MPa. The enzymatic activity of coated alpha-chymotrypsin complexes for dipeptides synthesis at 10.1 MPa in supercritical CO(2) (SC-CO(2)) was higher than that in a liquid CO(2) and ethyl acetate solution at 6.1 MPa. The behavior of alpha-chymotrypsin in SC-CO(2) was similar to that in liquid ethyl acetate. And increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in SC-CO(2). Furthermore, the control of the water content in the reaction media had a dominant effect on the enzymatic activity. The maximum conversion for the dipeptide synthesis by the surfactant-coated alpha-chymotrypsin was obtained at 4% water content. The alpha-chymotrypsin complexes exhibited a higher enzymatic activity than native alpha-chymotrypsin in SC-CO(2). The nonionic surfactants l-glutamic acid dialkyl ester ribitol amide and sorbitan monostearate were more favored than the anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate.     

Conformation and activity of lysozyme on binding to two types of gold nanorods: a comparative study

The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration.     

Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media

Structural ring-opening of lactones driven by enzymatic polymerization has been performed using low concentration dosages of surfactant-coated lipases in organic media. By comparison, enzymatic polymerization rate with coated lipase proceeded at a rate 100-fold better than native powder. Similarly a higher polymeric molecular weight (21,300), narrow dispersity (Mw/Mn=1.9) and better conversion (100%) were obtained following polyesterification tests with surfactant-coated lipase.     

A simple and rapid purification of commercial trypsin and chymotrypsin by reverse-phase high-performance liquid chromatography

Commercial trypsin and chymotrypsin were further purified with respective recoveries of approximately 80 and 50% of the activity in a reverse-phase high-performance liquid chromatography system using acetonitrile in dilute trifluoroacetic acid at pH 2. The purified enzymes showed single enzymatic activities toward synthetic and protein substrates. The enzymes can be rapidly purified in amounts appropriate for structural analysis of proteins.     

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.     

FORMATION OF NEW CRYSTALLINE ENZYMES FROM CHYMOTRYPSIN : ISOLATION OF BETA AND GAMMA CHYMOTRYPSIN

A solution of chymotrypsin on slight hydrolysis undergoes an irreversible change into new proteins, two of which are enzymes and have been isolated in crystalline form. The new crystalline enzymes, called beta and gamma chymotrypsins, differ from the original chymotrypsin as well as from each other in many physical and chemical respects, such as molecular weight, crystalline form, solubility, and combining capacity with acid. The new enzymes still possess the same enzymatic properties as chymotrypsin. It thus appears that the irreversible change from chymotrypsin to the new enzymes does not affect the structure responsible for the enzymatic activity of the molecule. The solubility curves of the new enzymes agree approximately with the curves for a solid phase of one component and furnish very good evidence that the preparations represent distinct substances. The various enzymes when mixed at the proper pH have a tendency to form mixed crystals of the solid solution type. Thus at pH 4.0 gamma chymotrypsin combines to form solid solution crystals with either alpha or beta chymotrypsin. Hence at this pH separation of gamma from either alpha or beta by means of fractional crystallization is impossible. At pH 5.0–6.0, however, each material crystallizes in its own characteristic form and at its own rate; thus a fractional separation of the various enzymes from each other becomes feasible.     

Single-molecule enzymology of chymotrypsin using water-in-oil emulsion

Single-molecule studies allow the study of subtle activity differences due to local folding in proteins, but are time consuming and difficult because only a few molecules are observed in one experiment. We developed an assay where we can simultaneously measure the activity of hundreds of individual molecules. The assay utilizes a synthetic chymotrypsin substrate that is nonfluorescent before cleavage by chymotrypsin, but is intensely fluorescent afterward. We encapsulated the enzyme and substrate in micron-sized droplets of water surrounded by silicone oil where each microdroplet contains <1 enzyme on average. A microscope and charge-coupled device camera are used to measure the fluorescence intensity of the same individual droplet over time. Based on these measurements, we conclude that enzymatic reactions could occur within this emulsion system, the statistical average activity of individual chymotrypsin molecules is similar to that measured in bulk, and the activity of individual chymotrypsin is heterogeneous.     

A new method to evaluate the thermal stability of lyophilized enzymes

Summary The thermal inactivation of lyophilized chymotrypsin was studied at controlled water activities. At 60 °C the enzyme showed good stability except at aw 0.97, whereas at 75 °C considerable inactivation occured at most water activities. Increasing the amount of buffer on the preparation decreased the stability significantly. The optimal temperature of enzymatic activity was increased 14 °C, when the water activity was decreased from 1 to 0.5.     

Enzymatic catalysis in nonaqueous solvents

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue.

The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.     

Interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.     

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.     

Proteasomal activity in mammalian spermatozoa

The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.     

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.     

Studies on the properties of hypothalamic luteinizing hormone-releasing hormone

The effects of various chemical and enzymatic treatments on the biological activity of porcine luteinizing hormone-releasing hormone (LH-RH) are described. This experiment was performed before the elucidation of the structure of LH-RH. LH-RH activity was abolished by the following endopeptidases: chymotrypsin, subtilisin, papain, and thermolysin, but not by pepsin or trypsin. Exopeptidases did not affect LH-RH activity, but a purified preparation of pyrolidone carbosylpeptidase did. The amino acid sequence of LH-RH/FSH-RH was established to be (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Amine. This decapeptide lacks both the Amine terminus and the COOH terminus. Its Amine-terminal dipeptide sequence,(pyro)Glu-His, is similar to that of tyrotropin-releasing hormone. The lack of inactivation by the exopeptidases is in good agreement with these findings. Treatment with various chemical reagents showed that tyrosine, histidine, tryptophan, and arginine in LH-RH are important for its biological activity. Nitrous acid and Edman degradation did not inactivate LH-RH. These results are also in agreement with the determined structure of LH-RH. This hormone showed a high follicle-stimulating hormone-releasing hormone (FSH-RH) activity. The inactivation of LH-RH was always accompanied by a loss of FSH-RH activity. These experiments also shed some light on the structure-activity relationship of this hormone.