A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.     

Stability of Glucose 6-Phosphate Dehydrogenase Complexed with Its Substrate or Cofactor in Aqueous and Micellar Environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), or cofactor, NADP⁺, has been studied within the range 20–40°C in three media: (a) 0.04 M NaOH–glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, k _in(s^–1). In the case of G6PDH–NADP⁺complexes, the values of k _inwere independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of k _infor the complex G6PDH–GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of k _incorresponded to the degree of hydration W ₀= 16.7; at W ₀> 16.7 and W ₀< 16.7, k _inincreased. Within the range 20–40°C, the values of k _inmeasured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of k _inwere characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6°C to 40°C. In all media studied, NADP⁺complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH–NADP⁺melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

Effect of trypsin microenvironment on the rate constants of elementary stages of the hydrolysis reaction of N α-Benzoyl-L-arginine ethyl ester

The hydrolysis reaction of N ^α-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant k _cat and the rate constant of the second order k _cat/K _m (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in reverse micelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k ₁. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.     

Reverse micelles: a novel tool for H2 production

Reverse micelles serve as a novel tool to entrap enzymes and microbial whole cells within aqueous pockets and can be of great use in enhancing the efficiency and sustainability of the biological system. Photosynthetic bacterium Rhodopseudomonas sphaeroides entrapped inside the aqueous pool of reverse micelles prepared from benzene-sodium lauryl sulphate exhibited 25-fold enhancement of H₂ photoproduction rate (1.67 ml H₂ [mg protein]^–1 h^–1) compared to cells suspended in normal aqueous medium. Hydrogen photoproduction by the bacterium was catalysed by the nitrogenase enzyme system which was supported at a low light intensity of 12 Em^–2 sec^–1 photon flux energy at a wavelength of 520 nm. The optimum temperature for the process was 40 °C.     

The phenomenon of super activity in dihydrofolate reductase entrapped inside reverse micelles in apolar solvents

Bovine liver dihydrofolate reductase has been solubilized in reverse micelles of cationic surfactant cetyltrimethylammonium bromide (CTAB) in isooctane-chloroform (1:1,V/V) mixture. Variation of waterpool (WO), pH and surfactant concentration showed that the enzyme activity was regulated by these parameters and was higher than the activity found in aqueous buffer (defined as superactivity); the maximum being at WO 13.3, pH 7.0 and CTAB concentration 75 mM. The Michaelis constants, Km for the substrate FAH2 and NADPH were found to be greater than those determined in water. Since reverse micelles have some features similar to those of biomembranes, display of super activity by dihydrofolate reductase indicates that enzymes in vivo may possess higher activity than actually observed in vitro studies in aqueous solutions.     

Stabilization of acid phosphatase in DDDACl/n-butyl acetate reverse micelles

Storage stability of acid phosphatase entrapped in reverse micelles was studied. Supramolecular systems were prepared with a cationic twin chain surfactant, didodecyldimethylammonium chloride (DDDAC1), n-butyl acetate as an organic solvent and different water percentages. The rate of enzyme deactivation was monitored in the temperature interval from 20 to 45?°C, at bulk pH from 4.8 to 6.4, either unstirred conditions or under convective mixing from 250 to 750 rev min^?1, water-to-surfactant molar ratio (w ₀) equal to 11.4, 12.7, 14.2 and with the following buffers, Na-citrate, Li-citrate, K-citrate, Na-propionate. Acid phosphatase entrapped in buffer pools of reverse micelles exhibited enhanced stability in comparison with the enzyme in the pure aqueous phase. Half-life was up to 4 times larger. Both the chemicals used for buffer preparation and buffer pH change, within one unit, were found to influence the rate of acid phosphatase deactivation. The activation energy of enzyme deactivation process in micellar systems was slightly increasing with w ₀ but the values were not very different from the one in aqueous phase (145.3?kJ?mol^?1). The rate of deactivation of enzyme confined in the micelles when shear stress was applied was reduced in comparison with that of the free protein, even though the percentage loss was greater.     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.     

Bounded water kinetic model of β-galactosidase in reverse micelles

In the present investigation, -galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (W_o) were collected. It was observed that the usual kinetic model used for -galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and W_o. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

反胶束萃取技术分离胰激肽原酶

研究了用十六烷基三甲基溴化铵（CTAB）/正己醇/正辛烷反胶束溶液萃取和反萃取商业用胰激肽原酶时,水相pH值、离子强度和种类、CTAB浓度和助表面活性剂浓度等因素对分离效率的影响,并从反胶束微观结构给予解释。结果表明：［CTAB］＝0.02 mol•L-1,正己醇／正辛烷(V/V)＝1：5,萃取pH＝9.0,反萃pH＝7.0,萃取［KBr］＝0.1 mol•L-1,反萃［KBr］＝1.5 mol•L-1,反萃取加15％乙醇(V/V)时,萃取率接近100%,反萃取活性回收得率在80%以上。商业用酶的纯化倍数最高为1.97倍,粗酶为7.15倍,且粗酶纯化后比活在200U／mg以上,电泳分析证实了纯化效果,显示了很好的工业前景。     

Enhanced enzymatic activity in reverse micelles

Summary The bell shaped dependence of the superactivity of enzymes solubilized in ionic reverse micelles (RMs) on the hydration ratio (W₀) is theoretically explained. The superactivity is due to enhanced concentration of the substrate (which has the same kind of charge as that of the surfactant head groups) near the enzyme surface. The opposing effects of the increase in the absolute charge of the surface of the RM and in the water pool width with W₀ cause a maximum in activity.     

Studies on Bacillus licheniformis endo-\-1,3-1,4-d-glucanase: characterization and kinetic analysis

Summary A new purification procedure for endo-\-1,3-1,4-d-glucanase from Bacillus licheniformis is described. The secreted enzyme was purified both from B. licheniformis and from recombinant Escherichia coli harbouring the cloned gene by ion exchange chromatography on a CM-Sepharose matrix at pH 5.6. The mature enzyme was resistant to proteolysis by trypsin and chymotrypsin but it was slowly digested by protease V8. It showed a continuous trimming where no large-limit polypeptides were noticeable thus supporting a monodomain structure. Former appearing peptides have been assigned theoretically according to the protein sequence and predictive methods of accessible areas. Kinetic parameters for the hydrolysis of barley \-glucan and lichenan by measuring the net release of reducing sugars at the optimum pH (7.02) and temperature (55° C) are k _cat=3500 ±800 s^–1 (turnover number) and K _m=1.45±0.21 mg/ml for barley \-glucan and k _cat=3000±750 s^–1 and K _m=1.98±0.40 mg/ml for lichenan. Correspondence to: E. Querol     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

A new acid carboxypeptidase,O-1, fromAspergillus oryzae

A new acid carboxypeptidase was purified fromAspergillus oryzae grown on solid bran culture medium. The purified enzyme was found to be homogeneous by disc gel electrophoresis at pH 9.4 and isoelectric focusing. The enzyme was termedA. oryzae acid carboxypeptidase O-1 with isoelectric point 4.08. The substrate specificity of the new enzyme was investigated with proangiotensin, angiotensin, and bradykinin. Even when the proline was present at the penultimate position of the peptide, the enzyme rapidly hydrolyzed the carboxyterminal Pro-X (X=amino acid) peptide bond. TheK _m andk _cat values for angiotension (–Pro⁷–Phe⁸) at pH 3.7 and 30°C were 0.2 mM and 1.7 sec^–1, respectively.