Estimation of Cellobiohydrolase I Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase I (CBH I),p-nitrophenyl β-D-cellobioside,p-nitrophenol andcellobiose show distinct ultraviolet spectra,allowing the design of an assay to track the dynamic process ofp-nitrophenyl β-D-cellobioside hydrolysis by CBH I.Based on the linear relationship between p-nitrophenolformation in the hydrolysate and its first derivative absorption Curve of AUC340_400_(nm)(area under the curve),a new sensitive assay for the determination of CBH I activity was developed.The dynamic parameters ofcatalysis reaction,such as Vm and k_(cat),can all be derived from this result.The influence of β-glucosidase andendoglucanase in crude enzyme sample on the assay was discussed in detail.This approach is useful foraccurate determination of the activity of CBHs.     

β-Glucosidase Catalyzing Specific Hydrolysis of an Iridoid β-Glucoside from Plumeria obtusa

An iridoid β-glucoside, namely plumieride coumarate glucoside, was isolated from the Plumeria obtusa （white frangipani） flower. A β-glucosidase, purified to homogeneity from P. obtusa, could hydrolyze plumieride coumarate glucoside to its corresponding β-O-coumarylplumieride. Plumeria β-glucosidase is a monomeric glycoprotein with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The purified β-glucosidase had an optimum pH of 5.5 for p-nitrophenol （pNP）-β-D-glucoside and for its natural substrate. The Km values for pNP-β-D-glucoside and Plumeria β-glucoside were 5.04±0.36 mM and 1.02±0.06 mM, respectively. The enzyme had higher hydrolytic activity towards pNP-β-D-fucoside than pNP-β-D-glucoside. No activity was found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity for the glucosyl group attached to the C-7＂ position of the coumaryl moiety of plumieride coumarate glucoside. The enzyme showed poor hydrolysis of 4-methylumbelliferyl-β-glucoside and esculin, and did not hydrolyze alkyl-β-glucosides, glucobioses, cyanogenic-β-glucosides, steroid β-glucosides, nor other iridoid β-glucosides. In conclusion, the Plumeria β-glucosidase shows high specificity for its natural substrate, plumieride coumarate glucoside.     

Structural changes of cellobiohydrolase I (1,4-β-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitro-phenyl-β-D-cellobioside) during the binding process

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demonstrated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.     

Alterations of cardiac and lymphocyte β-adrenoceptors in rat with chronic heart failure

The alterations of cardiac and lymphocyte β-adrenoceptors were observed in the rats with chronic heart failure produced by constriction of both abdominal aorta and renal artery. The results showed that β1-adrenocep-tor density and mRNA levels were increased, whereas these levels remained unchanged for β2 The concentration-contractile response curve for isoproterenol was shifted to the right in cardiac atrium, whereas the concentration-cAMP accumulation response curve for isoproterenol in myocardium was not changed. The number of β-adrenoceptors in blood lymphocyte was markedly reduced. Thus in the heart-failure rats the density of cardiac β-adrenoceptor was increased accompanying reduced β-adrenoceptor-mediated positive inotropic response, suggesting a post adenylate cyclase dys-function or impaired contractile components. In contrast, the alteration of β-adrenoceptor in lymphocyte is consistent with the reduced β-adrenoceptor-mediated inotropic response in heart.     

Structural changes of cellobiohydrolase I (1,4-beta-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitrophenyl-beta-D-cellobioside) during the binding process

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demon-strated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Effects of Gibberellic Acid on Primary Terpenoids and △^9 -Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell： the cytosolic mevalonic acid （MVA） pathway and the plastidial methylerythritol phosphate （MEP） pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid （GA3） on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase （DXS） and 3-hydroxy-3-methylglutaryl coenzyme A reductase （HMGR）, in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Ag-tetrahydrocannabinol （THC） contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Screening and molecular characterization of Serratia marcescens VITSD2: A strain producing optimum serratiopeptidase

The current work was attempted to isolate and characterize the serratiopeptidase producing Serratia sp. Among the 10 bacterial isolates 7 strains were identified as Serratia sp. Out of 7 strains one showed potent proteolytic activity and selected for further studies. Based on the morphological, biochemical and molecular characterization, the potent isolate （RH03） was identified as Serratia marcescens （GenBank accession number： KC961637） and the strain was designated as Serratia marcescens VITSD2. The production of serratiopeptidase was carried out in trypticase soya broth and the enzyme was partially purified using ammonium sulfate precipitation and dialysis. The specific activity was determined by casein hydrolysis assay and was found to be 12.00, 21.33, and 25.40 units/rag for crude, precipitated and dialysed samples. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 50 kDa. The antibacterial activity of the produced serratiopeptidase showed moderate activity against Pseudomonas aeruginosa MTCC No. 4676 （12 mm） and Escherichia coli MTCC No. 1588 （15 mm）.     

Expression of an endoglucanase from Tribolium castaneum （TcEG 1 ） in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation （saccharification） and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum （TcEG 1）, and evaluated the activity of the transgenic product （rTcEG 1）. Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi.

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p-nitrophenyl beta-D-cellobioside and p-nitrophenyl beta-D-lactosde as substrates was conducted: endoglucanase Z exhibited Km values of 3 mM and 7.5 mM and Vm values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (kcat = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (kcat = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and beta-cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl-enzyme intermediate.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

New substrate specificity of modified porcine pancreatic alpha-amylase

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.     

Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay for Cellobiohydrolase I

A double-antibody sandwich enzyme-linked immunosorbent assay was developed for quantifying cellobiohydrolase I (CBH I) in crude preparations of the cellulase complex from Trichoderma reesei. The other enzymes (endoglucanase and β-glucosidase) in this complex and other ingredients in culture broth did not interfere with this assay. The antibody configuration that resulted in the highest specificity for the assay of CBH I employed a monoclonal antibody to coat wells in polystyrene plates and peroxidase-labeled polyclonal antibody to detect cellobiohydrolase bound to the immobilized monoclonal antibody. Previously, procedures have not been available for the direct assay of CBH I activity in the presence of the other enzymes in the complex, and current indirect procedures are cumbersome and inaccurate. The direct procedure described here is highly specific for CBH I and useful for quantifying this enzyme in the range of 0.1 to 0.8 μg/ml.     

Synergistic cooperation of cellulases of trichoderma reesei as distinguished by a new filter-paper destruction assay

WHATMAN 1 CHR filter paper manufactured from macerated cotton fibers was shown to be a soft substrate when broken down by purified cellulases of Trichoderma reesei (CELLUCLAST). Destruction of filter-paper disks was induced by CBH I/1, CBH I/2, CBH II/1, CBH II/2, and EG I in a macroscopic assay. Attack on disks by mixtures of these cellulases (CBH I/1 or CBH I/2 mixed with CBH II/1, CBH II/2, or with EGJ) were followed by synergistically enhanced destructions. SCHLEICHER &SCHUELL filter paper No 595 was shown to be a harder substrate of enzymatical decomposition when induced by cellulases of CELLUCLAST. None of the cellulases could induce macroscopic destruction of filter-paper disks when acting in isolation. However, mixtures of isolated exo and endo-glucanases (CBH I/1 or CBH I/2 mixed with CBH II/1, CBH II/2, or EG I) caused powerful destruction of filter-paper disks. SCHLEICHER &SCHUELL filter paper No 595 incubated first with an endo-glucanase (CBH II/1, CBH II/2, EG I) and treated in a secondary incubation with an exo-glucanase (CBH I/1, CBH I/2) were destroyed to a greater extent than with incubations executed in the reverse order. Results confirm the endo exo concept of explaining cellulose decomposition. The filter-paper destruction assay was performed with filter-paper disks prepared with an office punch. Disks were incubated in 1 ml EPPENDORF reaction tubes filled up beforehand with 0.4 or 0.5 ml of enzyme solution. The degree of synergism of cellulases resulted from the assay in the range of 300 to 1 300 p.c.     

Isolation, characterization, and esterase and CO2 hydration activities of ubiquitin from bovine erythrocytes

Ubiquitin was isolated from bovine erythrocytes by a relatively simple procedure involving extraction with chloroform and ethanol, chromatography on DEAE-cellulose, and gel filtration. Amino acid and partial sequence analyses showed it to be identical to previously isolated material. Ubiquitin released p-nitrophenolate from p-nitrophenyl acetate, but did not cleave other esterase substrates that were tested. It had a turnover number of 116 mmol for p-nitrophenyl acetate at pH 7.7 and 30 degrees C, and this activity was relatively stable to heat treatment. Electrophoretic studies indicated that the ubiquitin was sequentially acetylated by p-nitrophenyl acetate, as judged by the appearance of more anodically migrating components. The reactions of ubiquitin with p-nitrophenyl acetate at pH 7.0 were biphasic and consisted of (a) an initial phase, during which the release of p-nitrophenol resulted from monoacetylation of the ubiquitin and from ubiquitin-catalyzed hydrolysis of the ester; and (b) a second phase, during which the release of p-nitrophenol resulted only from the breakdown and reformation of the acetyl-enzyme complex. Ubiquitin also showed CO2 hydration activity and could be localized following gel electrophoresis by the CO2-bromthymol blue staining method. The strong inhibitor of carbonic anhydrase, acetazolamide, also inhibited the CO2 hydration activity and p-nitrophenyl acetate activity of ubiquitin. An antibody against this protein did not precipitate bovine carbonic anhydrase II. The esterase activity of ubiquitin was much higher than those previously reported for the carbonic anhydrases.     

A monovalent anion affected multi-functional cellulase EGX from the mollusca,Ampullaria crossean

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.     

The titration of the active centers of cellobiohydrolase from Trichoderma reesei

A novel approach has been developed for the titration of enzyme active centers and for the determination of the molecular activity of enzymes. It is based on the simultaneous use of a nonspecific chromogenic substrate and a specific ligand (a substrate or an inhibitor), the latter being tightly bound with the enzyme's active center. The approach is demonstrated using the titration (that is, the determination of the molar concentration of the enzyme active centers) of purified cellobiohydrolase I (CBH I) (EC 3.2.1.91) of the fungus Trichoderma reesei. p-Nitrophenyl-beta-D-lactoside was used as a reference substrate (Km = 0.5 mM), and cellobiose and CM-cellulose as specific ligands. The molecular weight of CBH I as it was determined by the titration with cellobiose was 42,000 +/- 3,000. The inhibition constant by cellobiose was (6 +/- 1) X 10(-6) M. The value of the catalytic constant for the hydrolysis of p-nitrophenyl-beta-D-lactoside calculated from the titration data was equal to 0.063 s-1. CM-cellulose turned out to be more efficient titration agent for cellobiohydrolase than cellobiose, and might be used for the titration of the enzyme in concentrations of the latter of 0.008-0.02 mg/ml. The titration data showed that the inhibition constant of CM-cellulose toward CBH I was equal to (1.0 +/- 0.2) X 10(-7) M.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.