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A novel esterase showing activity specific for esters of aryl-carboxylic acids was discovered in Sporosarcina sp. nov., which was identified by the 16S rDNA sequencing method in addition to morphological and physiological analyses. The aryl-carboxylesterase (named EstAC) was purified 780-fold from crude cell extracts by a 5-step procedure. EstAC was characterized as a monomeric protein with a molecular weight of 43,000, an optimum pH of around 9.0, and an optimum temperature of 40 °C. The pH optimum and the effects of inhibitors together with an internal amino acid sequence suggested that EstAC is a member of family VIII esterases. EstAC was found to be highly active on a wide variety of substrates such as alkyl benzoates, alkyl phenylacetates, ethyl α- or β-substituted phenylpropionates, dialkyl terephthalates, dimethyl isophthalate, and ethylene glycol dibenzoate. However, monomethyl terephthalate was not hydrolyzed. It was suggested that EstAC had 4-hydroxybenzoyl and cinnamoyl esterase activities as well.

Poly(epsilon-L-lysine) (epsilon-PL) is a naturally-occurring L-lysine homopolymer having antimicrobial activity. A newly-isolated strain of Streptomyces aureofaciens produced a short chain length epsilon-PL consisting of 5-20 residues at the highest production level of 4.5 g l(-1). This epsilon-PL had different spectra in terms of antimicrobial activity from the epsilon-PL that is now used as a food preservative. The high productivity was based on multiple metabolic pathways for L-lysine synthesis, and a great flux from L-lysine to epsilon-PL. The usefulness of this new epsilon-PL and its producing strain was discussed.

Two poly(epsilon-L-lysine) (epsilon-PL) producer strains of Streptomyces celluloflavus secreted a novel polymeric substance into their culture broths along with epsilon-PL. Three types of HPLC analysis plus one- and two-dimensional 1H and 13C nuclear magnetic resonance experiments revealed that the secreted substance was poly(gamma-L-diaminobutanoic acid) (gamma-PAB), an L-alpha,gamma-diaminobutanoic acid (L-DAB) homopolymer linking between y-amino and alpha-carboxylic acid functional groups. The gamma-PABs from the two strains had an identical chemical structure, and the same number-average molecular weight of 2100-2200. No copolymers composed of the two amino acids L-DAB and L-lysine were found in either of the broths from the producers. Both strains coproduced high levels of the two poly(amino acid)s in the presence of SO4(2-) at pH 4.0 and 4.5 L min(-1) aeration in a 5-L jar fermentor. gamma-PAB exhibited strong inhibitory activities against various yeasts and weaker actions against bacteria than epsilon-PL. gamma-PAB may have various biological functions similar to epsilon-PL, and the use of gamma-PAB along with epsilon-PL would be advantageous for technical applications in various fields.

Poly(epsilon-L-lysine) (epsilon-PL) is a naturally occurring poly(amino acid) characterized by a unique structure linking epsilon-amino and carboxyl groups of L-lysine. Due to its various functions and its biodegradability and non-toxicity, the epsilon-PL polymer has attracted increasing attention in recent years. epsilon-PL is frequently found in various strains of Streptomyces sp. This review gives an up-to-date overview regarding the biosynthesis of epsilon-PL focussing mainly on results obtained from ten newly isolated producer strains, using the two-stage culture method of cell growth and epsilon-PL production cultures. The production of nearly monodispersed epsilon-PL is covered together with the development of epsilon-PL specific hydrolases and the release of synthesized epsilon-PL into the culture broth. From these results, coupled with the termination of polymerization through nucleophilic chain transfer, the biosynthetic mechanism of the polymer is discussed.

The presence of poly(epsilon-L-lysine) (epsilon-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained epsilon-PLs, when they were produced from glucose, showed a polydispersity index of Mw/Mn = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the Mn and slight increases in the Mw/Mn. These observations indicated the chain length and polydispersity of epsilon-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that SO4(2-) was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of epsilon-PL.

The biosynthesis of poly(epsilon-L-lysine) (epsilon-PL) in the two newly isolated strains of Streptomyces lydicus USE-11 (USE-11) and Streptomyces sp. USE-51 (USE-51) was studied by a newly developed two-stage culture method of cell growth at pH 6.8 and epsilon-PL production at pH 4.5. USE-11 synthesized epsilon-PL consisting of about 28 residues at a high production level, whereas USE-51 did the polymer with 15 ones at a low level. The secreted epsilon-PLs in culture media were digested in a neutral pH range with a peptide hydrolase(s) produced by the epsilon-PL producers. The optimum production levels were presumed to be dependent upon the inherent epsilon-PL synthesis machinery of each producer. The production in USE-51 was sharply dependent upon cell density as was often observed in the production of antibiotics, whereas that in USE-11 was scarcely affected by the density. The SO(4)(2-) was found to be essential for the epsilon-PL production in both strains. This might suggest the involvement of a thiol group in the polymerization reactions including the activation of L-lysine. This study indicates that USE-11 is a most suitable strain for the exploration of the epsilon-PL biosynthesis at the molecular level as well as for the technical applications.

Background: Partial resection of the posteromedial aspect of the olecranon in the treatment of valgus extension impingement osteophytosis is a well-described technique. It has been hypothesized that removal of the normal olecranon process, beyond the osteophytic margin, increases the strain in the anterior bundle of the medial collateral ligament., Methods: We used an electromagnetic tracking device to investigate the strain in the anterior bundle of the medial collateral ligament as a function of increasing applied torque and posteromedial resections of the olecranon in seven cadaveric elbows. Applied torques under valgus stress consisted of hand weight, hand weight plus 1.75 Nm, and hand weight plus 3.5 Nm. Resections were conducted in sequential 3-mm increments, from 0 to 9 mm. We measured changes in the length of the anterior and posterior bands of the anterior bundle of the medial collateral ligament with strain gauges. The strains of the two bands were averaged, and the average was reported., Results: The strain in the anterior bundle of the medial collateral ligament was found to increase with increasing flexion angle, valgus torque, and olecranon resection beyond 3 mm. In two elbows, the anterior bundle of the medial collateral ligament ruptured during testing following the 9-mm resection. There was a significant difference between the strain following the 6-mm resection and that following the 3-mm resection at 110 degrees of flexion with 3.5 Nm of added torque (p = 0.004)., Conclusions: In this in vitro cadaver study, an increase in flexion angle, an increase in valgus torque, and resection of > or =6 mm led to an increase in strain in the anterior bundle of the medial collateral ligament. The non-uniform change in strain related to 3 mm of resection suggests that resections of the posteromedial aspect of the olecranon of >3 mm may jeopardize the function of the anterior bundle.

A new diterpene, dilkamural (1), of the secospatane class has been isolated from the brown alga Dilophus okamurae Dawson, and its structure, including absolute stereochemistry, has been determined by NMR spectroscopic and chemical means.

Efficient biochemical processes were developed for the synthesis of several chiral alcohol and acid intermediates of insecticides by a combination of strictly stereoselective hydrolytic enzyme-catalyzed reactions and subsequent chemical transformations with inversion or racemization of the chiral center of the undesired antipodes. The whole amounts of starting racemic mixtures are converted to desired stereoisomers in the processes, which are generally applicable to the industrial productions of various chiral secondary alcohols and alpha-substituted acids once a highly stereospecific enzyme is obtained for the target compounds. The alcohols reported here are: 1-(4-phenoxyphenoxy)-2-propanol, 1; 4-hydroxy-3-methyl-2-(2'-propynyl)-2-cyclopentenone, 2; and alpha-cyano-3-phenoxybenzyl alcohol, 3. The acids are 2, 2-dimethyl-3-(2-methyl-1-propenyl)-cyclopropanecarboxylic acid (chrysanthemic acid), 4; and 2-(4-chlorophenyl)-3-methylbutyric acid, 5. In addition, the mechanism of action of Pseudomonas cepacia lipase (PCL), the most effective enzyme for the resolution of 1, and the recombinant Arthrobacter globiformis esterase (AES) for 4 was studied from the reaction kinetics. The site-directed mutagenesis techniques were also used for AES. The results indicated that the stereoselectivity of PCL is caused by the position and direction of a medium-sized substituent at the chiral center of the alcohol moiety in the rate-determining breakdown of a tetrahedral intermediate in the acylation of the enzyme and that the catalytic site of AES has the characteristics of the penicillin-recognizing enzymes in which Ser 59 in the concensus motif Ser-X-X-Lys plays a vital role as a nucleophile during acylation and Lys 62 acts as a general base.

The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.

Dimeric alpha-chymotrypsin (D) formed over a range of low pH (3.7-5.2) showed a different specificity from monomeric alpha-chymotrypsin (M) for the acylation step, though the catalytic system functions as well in the dimeric enzyme as in the monomeric enzyme. For aliphatic substituted phenyl esters a bulky substituent at the ortho-position of the phenyl ring reduced the acylation rate of the dimeric enzyme compared with the monomeric enzyme. Especially for aliphatic p-nitrophenyl esters, the dissociation constants of the DS complexes were always smaller than those of the MS complexes, independent of the aliphatic chain length of the substrate. The intrinsic acylation rate constant of the dimeric enzyme increased with the chain length of the substrate. These catalytic properties are discussed with reference to the structures of the catalytic and binding sites of the dimeric enzyme.

The pH dependences of kcat/Km, kcat and Km of thermolysin-catalyzed hydrolysis of N-furylacryloylglycyl-L-leucinamide (Fua-Gly-LeuNH2) and N-furylacryloylglycyl-L-phenylalaninamide (Fua-Gly-PheNH2) were investigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell-shaped curve with pKa1 = 5.0 and pKa2 = 8.25, at 25 degrees C. Both kcat and Km increased with pH at lower pH and took larger values for Fua-Gly-LeuNH2 than for Fua-Gly-PheNH2 at 25 degrees C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy-L-phenylalanine and L-phenylalanyl-L-leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH. Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non-linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated delta H values of deprotonation for pKa1 and pKa2 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKa1 is too large to be assigned to the carboxylic group of Glu-143, in contrast to the generally accepted view. A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc-coordinated water.