The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016.

Postoperative atrial fibrillation in patients undergoing coronary artery bypass grafting or cardiac valve surgery: intraoperative use of landiolol.

BACKGROUND: Landiolol hydrochloride is a new ß-adrenergic blocker with a pharmacological profile that suggests it can be administered safely to patients who have sinus tachycardia or tachyarrhythmia and who require heart rate reduction. This study aimed to investigate whether intraoperative administration of landiolol could reduce the incidence of atrial fibrillation (AF) after cardiac surgery. METHODS: Of the 200 consecutive patients whose records could be retrieved between October 2006 and September 2007, we retrospectively reviewed a total of 105 patients who met the inclusion criteria: no previous permanent/persistent AF, no permanent pacemaker, no renal insufficiency requiring dialysis, and no reactive airway disease, etc. Landiolol infusion was started after surgery had commenced, at an infusion rate of 1 µg/kg/min, titrated upward in 3-5 µg/kg/min increments. The patients were divided into 2 groups: those who received intraoperative ß-blocker therapy with landiolol (landiolol group) and those who did not receive any ß-blockers during surgery (control group). An unpaired t test and Fisher's exact test were used to compare between-group differences in mean values and categorical data, respectively. RESULTS: Seventeen of the 105 patients (16.2%) developed postoperative atrial fibrillation: 5/57 (8.8%) in the landiolol group and 12/48 (25%) in the control group. There was a significant difference between the two groups (P=0.03). The incidence of AF after valve surgery and off-pump coronary artery bypass grafting was lower in the landiolol group, although the difference between the groups was not statistically significant. CONCLUSIONS: Our retrospective review demonstrated a marked reduction of postoperative AF in those who received landiolol intraoperatively. A prospective study of intraoperative landiolol for preventing postoperative atrial fibrillation is warranted.

On the preparation of indoxyl red from indican and some new characteristics.

An indole compound with a strong purple-red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, (13)C NMR, (1)H NMR, UV-visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel(9) 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202.

A case of cancer pain management by long-term intrathecal PCA.

Titration of oral or intravenous medication is the preferred method of pain management for most patients with cancer pain. However, some patients experience insufficient pain relief or considerable adverse effects from systemic opioids. For these reasons, the control of severe cancer pain continues to present a variety of challenges to clinicians. We report our experience of successfully managing cancer pain in a patient by means of long-term intrathecal administration of morphine, bupivacaine, and racemic ketamine via a patient-controlled delivery system. This therapy reduced the patient's nausea, vomiting, and somnolence, led to early hospital discharge, and increased her level of daily activity. There were no signs of motor paralysis, psychomimetic alteration, neurological dysfunction, or infection related to the intrathecal route during treatment. Intrathecal therapy is an effective treatment in terminally ill patients.

Purification, characterization, molecular cloning, and expression of a new aminoacylase from Streptomyces mobaraensis that can hydrolyze N-(middle/long)-chain-fatty-acyl-L-amino acids as well as N-short-chain-acyl-L-amino acids.

We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).

Temperature scanning FTIR analysis of interactions between sugar and polymer additive in amorphous sugar-polymer mixtures.

The impact of a polymer additive (polyvinylpyrrolidone, PVP) on hydrogen bonding in amorphous sugar matrices as well as on the glass transition temperature, T(g), were examined by temperature scanning Fourier transform infrared spectroscopy (TS-FTIR). An amorphous sugar matrix containing PVP was prepared by air-drying an aqueous solution of a sugar-PVP mixture. The hydrogen bonds in the sugar-PVP mixture (sugar-PVP and sugar-sugar hydrogen bonds) were analyzed from the IR peak positions corresponding to the stretching vibration of C==O groups of PVP and O--H groups of the sugar and the temperature dependence of the peak position of the O--H stretching vibration band. The addition of PVP to amorphous mono and disaccharides significantly lowered the extent of hydrogen bond formation while interactions between sugars and the PVP tended to prevent the disruption of hydrogen bonds due to increasing temperature, the magnitude of which was larger for larger oligomers. The T(g) value for the amorphous sugar was increased by the addition of PVP in many cases. As the size of sugar molecule became larger, the relative magnitude of the increased T(g) by PVP to the difference between the T(g) values for sugar alone and PVP alone became larger and then reached a certain level; it was slight in the case of glucose. Collectively, these results demonstrate that the magnitude of the impact of PVP on an amorphous sugar matrix strongly vary and are dependent on the types of sugar.

Repair of an infrarenal abdominal aortic aneurysm is associated with persistent left ventricular diastolic dysfunction.

BACKGROUND: Left ventricular (LV) diastolic function has received much attention recently. However, few studies have evaluated LV diastolic function in the perioperative period. The aim of this study was to elucidate perioperative changes in diastolic function using tissue Doppler imaging (TDI) in patients undergoing repair of an infrarenal abdominal aortic aneurysm (AAA). METHODS: Eight patients undergoing repair of an infrarenal AAA were studied prospectively using transesophageal echocardiography. Doppler echocardiographic examinations were performed before the surgical procedure (T1), immediately before aortic unclamping (T2), 30 minutes after aortic unclamping (T3), and at the end of surgery (T4). RESULTS: Pulmonary edema developed in two patients on postoperative day 1. These two patients had the lowest early diastolic mitral annular velocity (Ea) of the study group at the end of surgery. The ratio of the peak velocity of early mitral inflow (E) to the peak velocity of atrial inflow was significantly decreased at T3 and T4. The systolic ejection velocity was significantly decreased at T3, but returned to the baseline value at T4. The Ea was significantly decreased at T3 and T4. The E/Ea ratio showed a progressive rise and was significantly increased at T3 and T4. CONCLUSIONS: In patients undergoing repair of an infrarenal AAA, the Ea derived using TDI decreases at T3 and is still reduced at T4. The E/Ea ratio, which is used to estimate LV filling pressures, is significantly increased at T3 and T4. Further research is required to confirm the development of diastolic dysfunction and determine its possible association with increased postoperative morbidity and mortality.

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes.

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.

Large volume loading to prevent cisplatin-induced nephrotoxicity during negative-balance isolated pelvic perfusion.

PURPOSE: Negative-balance isolated pelvic perfusion (NIPP) is used to administer high doses of anticancer drugs such as cisplatin to patients with advanced cancer of the pelvic region. Although the drugs are intended to be specifically delivered to the pelvis, their leakage into the systemic circulation can cause acute renal failure. This study examines the loading volume required for preservation of renal function during anesthesia of NIPP. METHODS: Pelvic cancer patients were assigned to NIPP according to its enrollment criteria. Patients with heart failure, uncontrollable hypertension, renal failure, pulmonary disease or contraindication for the contrast media were excluded. We compared the current anesthesia management regime with a previous protocol, with regard to the loading volume and renal function as assessed by the calculated glomerular filtration rate (GFR). The correlation between the total loading volume and the GFR ratio (GFR after NIPP/GFR before NIPP) was evaluated to define adequate volume loading. RESULTS: The GFR ratios were 0.86 +/- 0.29 and 1.12 +/- 0.25 for the previous and current procedures, respectively. The regression line showed that a minimum loading volume of 28.8 ml kg(-1) h(-1) was required to maintain a GFR ratio of > or =1. CONCLUSIONS: A large volume infusion preserves the GFR despite high-dose cisplatin administration by NIPP.

TPR domain of Ser/Thr phosphatase of Aspergillus oryzae shows no auto-inhibitory effect on the dephosphorylation activity.

A Ser/Thr phosphatase gene cloned from Aspergillus oryzae, aoppt, revealed that the tetratricopeptide repeat (TPR) and catalytic domains of the full-length AoPPT are located at the N- and C-terminal regions, respectively, similar to those of human Ser/Thr phosphatase 5 (PP5) and yeast Ppt1. Four different regions of AoPPT, namely, a full-length polypeptide, the catalytic domain, the catalytic domain plus C-terminal 15 amino-acid residues and the TPR domain were expressed in Escherichia coli and their roles in dephosphorylation activity were examined, using p-nitrophenyl phosphate as the substrate. The full-length AoPPT showed the highest dephosphorylation activity while the catalytic domain had the lowest activity. The activity of the catalytic domain was not inhibited by the presence of the TPR domain and arachidonic acid did not increase the activity of the full-length enzyme. These findings suggest that the integrity of the entire enzyme would be necessary for its full activity to be expressed.

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis.

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.

Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate.

A sandwich ELISA method using peptide tags showing a specific affinity to a hydrophilic polystyrene surface (PS-tags), PS 19 composed of RAFIASRRIKRP and KPS19R10 of KRAFIASRRIRRP and a hydrophilic polystyrene (phi-PS) plate was used to analyze protein-protein interactions. An Escherichia coli cysteine synthase complex, in which serine acetyltransferase (SAT) interacts with O-acetylserine sulfhydrylase-A (OASS) was used as a model system. When the interaction was detected by the conventional sandwich ELISA method using a hydrophobic polystyrene (pho-PS) plate, for the exclusive use of ELISA, the signal intensity was barely detectable due to conformational change of the ligand protein, OASS in the adsorbed state. On the contrary, when OASS, genetically fused with PS19 (OASS-PS19) or chemically conjugated with KPS19R10 (OASS-KPS19R10), was immobilized on the phi-PS plate, a high signal intensity was detected. Furthermore, by applying the two-step sandwich ELISA, in which OASS-PS19 or OASS-KPS19R10 formed a complex with SAT in the blocking solution before immobilization on the phi-PS plate, the signal intensity was further increased with a much shorter operational time, because SAT in the blocking solution formed a complex with OASS-PS19 or OASS-KPS19R10 without any steric hindrance.

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface.

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.

Effects of the supporting electrolyte on the kinetics of the removal of proteins adsorbed on a stainless steel surface by H2O2-electrolysis.

The effects of different types of supporting electrolytes on the removal of beta-lactoglobulin (beta-Lg) after being adsorbed to a stainless steel surface by a H2O2-electrolysis treatment was investigated. In this process, hydroxyl radicals (*OH), generated by the electrolysis of hydrogen peroxide, decompose the substances adhering to the surface. The removal of the adsorbed protein from the stainless steel surface during the treatment was monitored in situ by ellipsometry. The apparent first-order removal rate constants, k(cl), for 17 types of supporting electrolytes were determined, as well as the current corresponding to the rate of generation of *OH. The k(cl) and generated current values for LiCl, NaCl, KCl, KNO(3), K(2)SO(4), CH(3)COOK, and K(2)CO(3) were all similar. Ca(2+) and Mg(2+) strongly suppressed the removal of the adsorbed protein. The presence of ammonium compounds led to an increase in k(cl) and current values. In H2O2-electrolysis in the presence of potassium phosphate, the removal was extremely rapid, and an apparent increase in the thickness of the adsorbed layer was observed. The mechanisms responsible for the peculiar effects of calcium, magnesium, phosphate, and ammonium compounds were investigated by means of a Fourier transform infrared (FTIR) spectroscopic analysis, as well as by the characteristics of the removal under different treatment conditions.

Protease susceptibility of beta-lactoglobulin adsorbed on stainless steel surface as evidence of contribution of its specific segment to adsorption.

beta-Lactoglobulin (beta-Lg) is a major constituent of fouling deposits in the dairy industry. To determine the interaction between beta-Lg and stainless steel surfaces, beta-Lg irreversibly adsorbed on stainless steel particles was subjected to lysyl endopeptidase treatment and the course of fragmentation was compared with that observed for beta-Lg in solution. The results showed a distinct difference between the courses of fragmentation: a fragment (residues 102-135) was liberated readily from beta-Lg in solution but scarcely from beta-Lg irreversibly adsorbed on stainless steel particles. This result strongly suggests that residues 102-135 include a segment primarily responsible for the interaction of beta-Lg with stainless steel surfaces. This supports our previous results [Sakiyama et al., J. Biosci. Bioeng., 88, 536-541 (1999)] that showed that residues 125-135 of beta-Lg have a strong affinity toward stainless steel surfaces and probably a major contribution to the adsorption of beta-Lg.

Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins.

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli.

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.

Effects of ulinastatin treatment on the cardiopulmonary bypass-induced hemodynamic instability and pulmonary dysfunction.

OBJECTIVE: To examine the association between decreased release of proinflammatory cytokines in response to urinary trypsin inhibitor pretreatment and decreased myocardial and lung injury after cardiopulmonary bypass. DESIGN: A prospective, randomized, double-blind study. SETTING: University hospital. SUBJECTS: Thirty patients on cardiopulmonary bypass undergoing coronary artery bypass grafting. INTERVENTIONS: Patients received 5000 units/kg intravenous urinary trypsin inhibitor (n = 15) or 0.9% saline (control, n = 15) immediately before aortic cannulation for cardiopulmonary bypass. MEASUREMENT AND MAIN RESULTS: Neutrophil elastase, tumor necrosis factor-alpha, interleukin-6, and interleukin-8 were measured after intubation (T1), immediately before aortic cannulation (T2), after separation from cardiopulmonary bypass (T3), at the end of surgery (T4), and on postoperative days 1 (T5), 3 (T6), and 5 (T7). Simultaneous hematocrit values were obtained at all sample times. Isoenzyme of creatine kinase with muscle and brain subunits, troponin-T, and myosin light chain I were also measured. Various hemodynamic and pulmonary data were obtained perioperatively. Levels of neutrophil elastase and cytokines were corrected for hemodilution. Interleukin-6 and interleukin-8 levels were lower at T3 and T4 in the urinary trypsin inhibitor group than in the control group. Stroke volume index was significantly decreased in the control group at T3, and statistical difference was found between groups at T3 (p < .01). Respiratory index and intrapulmonary shunt were significantly higher in the control group than in the urinary trypsin inhibitor group at T3. Changes in respiratory index and intrapulmonary shunt correlated with interleukin-8 levels at T3 (r = .52, p < 00001; r = .37, p < 0001, respectively) and T4 (r = .44, p < .001; r = .24, p < .05, respectively). Neutrophil elastase levels and cardiac marker responses to coronary artery bypass grafting surgery were similar in both groups. CONCLUSIONS: Prepump administration of urinary trypsin inhibitor attenuates the elevation of interleukin-6 and interleukin-8 release immediately after cardiopulmonary bypass.

On the interaction site of serine acetyltransferase in the cysteine synthase complex from Escherichia coli.

Cysteine synthase from Escherichia coli is a bienzyme complex comprised of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase A. The site of interaction of a SAT molecule was investigated by gel chromatography and surface plasmon technique using various mutant-type SATs, to better understand the mechanism involved in complex formation. The C-terminus of SAT, Ile 273, along with Glu 268 and Asp 271, was found to be essential for complex formation. The effects of O-acetyl-L-serine and sulfide on the affinity for the complex formation were also studied using a surface plasmon technique.

A novel acylase from Streptomyces mobaraensis that efficiently catalyzes hydrolysis/synthesis of capsaicins as well as N-acyl-L-amino acids and N-acyl-peptides.

A novel enzyme that catalyzes efficient hydrolysis of capsaicin (8-methyl-N-vanillyl-6-nonenamide) was isolated from the culture broth of Streptomyces mobaraensis. The enzyme consisted of two dissimilar subunits with molecular masses of 61 and 19 kDa. The enzyme was activated and stabilized in the presence of Co2+. It showed a pH optimum of about 8 and was stable at temperatures of up to 55 degrees C for 1 h at pH 7.8. The specific activity of the enzyme for the hydrolysis of capsaicin was 10(2)-10(4) times higher than those for the enzymes reported to date. In an aqueous/n-hexane biphasic system, capsaicin analogues such as octanoyl, decanoyl, and lauroyl vanillylamides were synthesized from the corresponding fatty acids and vanillylamine at yields of 50% or greater. In addition, the enzyme catalyzed the deacylation of N-lauroyl-L-amino acids and N-lauroyl-L-dipeptides and the efficient synthesis of Nalpha-lauroyl-L-lysine, Nepsilon-lauroyl-L-lysine, and various N-lauroyl-peptides in aqueous solution in both the absence and the presence of glycerol.