Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-l-aspartic acid with l-phenylalanine methyl ester to form N-benzyloxycarbonyl-l-aspartyl-l-phenylalanine methyl ester (Z · Asp · Phe · OMe), the precursor to the sweet-tasting compound l-aspartyl-l-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-l-glycyl-l-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z · Asp · Phe · OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Effect of the ΔPhe residue configuration on a didehydropeptides conformation: A combined CD and NMR study

Conformations of two pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐OMe ( Z‐ OMe ), Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐OMe ( E‐ OMe ), Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐p‐NA ( Z‐p‐ NA ), and Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐p‐NA ( E‐p‐ NA ) were compared on the basis of CD and NMR studies in MeOH, trifluoroethanol (TFE), MeCN, chloroform, and dimethylsulfoxide (DMSO). The CD results were used as the additional input data for the NMR‐based determination of the detailed solution conformations of the peptides. It was found that E‐ OMe is unordered and Z‐ OMe , Z‐p‐ NA , and E‐p‐ NA adopt the β‐turn conformation. There are two overlapping β‐turns in each of those peptides: type II and type III′ in Z‐ OMe and Z‐p‐ NA , and two type III in E‐p‐ NA . The ordered structure‐inducing properties of Δ^ZPhe and Δ^EPhe in the peptides studied depend on the C‐terminal blocking group. In methyl esters, the Δ^ZPhe residue is a strong inducer of ordered conformations whereas the Δ^EPhe one has no such properties. In p‐nitroanilides, both isomers of ΔPhe cause the peptides to adopt ordered structures to a similar extent. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1055–1064, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Total enzymatic synthesis of cholecystokinin CCK-5

Summary. This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH₂ of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt·HCl by -chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester.The other fragment H-Asp(OMe)-Phe-NH₂ resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH₂·HCl and thermolysin as catalyst, followed by catalytic hydrogenation.Finally PhAc-Gly-Trp-Met-Asp-Phe-NH₂ was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH₂ with -chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the -methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Resolution of DL-2-aminosuberic acid via protease-catalyzed ester hydrolysis

Summary Papain-catalyzed regioselective cleavage of-methyl ester in Z-DL-Asu(OMe)-OMe leads to Z-L-Asu(OMe)-OH and Z-D-Asu(OMe)-OMe. Subsequent saponifications yield Z-L-Asu-OH and Z-D-Asu-OH. The enzymatic-ester hydrolysis was also achieved by subtilisin BPN in organic solvent with low water content.Abbreviations Asu 2-aminosuberic acid - Z benzyloxycarbonyl - OMe methyl ester - DCHA dicyclohexylamine     

A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides

A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase.

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8).     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.     

Synthesis of L-tyrosine glyceryl ester catalyzed by α-chymotrypsin in water-miscible organic solvents: A possible sun-tan accelerator product

Summary The synthesis of L-tyrosine glyceryl ester, from glycerol and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by -chymotrypsin. Values of 60 % (v/v) for glycerol and 200 mM for L-tyrosine methyl ester were optimal for the transesterification reaction. Additionally to glycerol, several other water miscible cosolvents (acetonitrile, N,N'-dimetyl formamide and tetrahydrofurane) were tested in the reaction media, but their presence did not give an enhancement on the transesterification activity with respect to the glycerol/water medium. However, increasing the hydrophobicity of the cosolvent resulted in a reduction of the enzyme activity, the water:glycerol mixture being the best reaction media.     

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.     

Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues.

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity

Thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na(+), K(+), Cl(-) and Br(-) in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) [Inouye, K. (1992) J. Biochem. 112, 335-340]. However, effect of divalent salts on thermolysin activity has not been investigated systematically. In this study, effect of Co(2+) ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3-4 times with increasing the Co(2+) concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co(2+) concentration (2-18 mM). The activation-and-inhibition dual effects of Co(2+) ion were analysed kinetically. Release of the catalytic Zn(2+) ion from thermolysin, concomitantly occurred with the Co(2+)-dependent activation, was measured with a Zn(2+)-specific fluorescent probe. This indicates that the activation is caused by substituting Co(2+) ion for the catalytic Zn(2+) ion. Meanwhile, the Co(2+)-dependent activation was inhibited competitively by Zn(2+) ion (0.1-1.0 muM) added, similarly to that it is inhibited by higher concentration of Co(2+) ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co(2+) and Zn(2+) ions.     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

Synthesis of aspartame precursor: alpha-L-aspartyl-L-phenylalanine methyl ester in ethyl acetate using thermolysin entrapped in polyurethane

Cross-linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z-L-aspartic acid (ZA) was reacted with -Lphenylalanine methyl ester (L-PM) in water-saturated ethyl acetate to give only alpha-Z-L-aspartylL-phenylalanine methyl ester (alpha-ZAPM). Ninety-four percent conversion of alpha-ZAPM was obtained for 30 h of reaction at 40 degrees C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU-ll-G-3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin-catalyzed condensation reaction of ZA with L-PM in water-saturated ethyl acetate was found to be first order in L-PM and the Lineweaver-Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L-PM with the immobilized enzyme and with the ZA-immobilized enzyme complex, with the second reaction being the rate determining step.     

Fatty acid composition of seeds of Satureja thymbra and S. cuneifolia

The chemical composition of fatty acid methyl esters (FAMEs) from seeds of S. thymbra and S. cuneifolia were analyzed by GC/MS. 7 FAMEs were identified from the seeds of S. thymbra mainly as 9-octadecenoic acid methyl ester (43.9%), hexadecanoic acid methyl ester (11.4%), 9,12,15-octadecatrienoic acid methyl ester (Z,Z,Z) (30.2%), and octadecanoic acid methyl ester (14.1%), while from the seed of S. cuneifolia 10 FAMEs were obtained with the main components, similar to S. thymbra. These were identified as 9-octadecenoic acid methyl ester (10.1%), hexadecanoic acid methyl ester (methyl palmitate, 34.6%), 9,12,15-octadecatrienoic acid methyl ester (Z,Z,Z) (6.3%) and octadecanoic acid methyl ester (1.8%).     

Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k_cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K_m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.     

(10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester as an anti-inflammatory compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E, 12Z, 15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-Otetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 microg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13hydroxy-9,11-octadecadienoic acid (5) and (9Z,llE)13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE500 microg of 63% and 79%, respectively). Compounds 1, 4 ((9Z, 12Z, 14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 microg/ml.