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.     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer

A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m. This preparation gave only one band by disc gel electrophoresis. Glucose was the only product of dextran hydrolysis. Optimum pH and temperature for the activity of the enzyme were pH 4.5–5.0 and 45°C, respectively. The enzyme was most stable over a pH range of 4.5–6.0, and after 2 hours at 50°C maintained over 60% of its original activity. The molecular weight was 29,000 daltons and the isoelectric point was at pH 7. K_m (45°C, pH 5) for dextran T-40 was 1.2×10^–5 M. Glucose inhibited the enzyme competitively with a K_i (45°C, pH 5) of 0.5 mM.     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.     

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.     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg²⁺ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 °C. The apparent K _m value for 4-chlorobenzoyl-coenzyme A was 2.4–2.7 µM. V _max was 1.1 × 10^–7 M sec^–1 (2.2 µmol min^–1 mg^–1 of protein). The NH₂-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.Abbreviations CBA chlorobenzoate - CoA coenzyme A - HBA hydroxybenzoate - DTT dithiothreitol - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis     

Production,purification and partial characterization of an endopolygalacturonase fromCryptococcus albidus var.albidus

Cryptococcus albidus var.albidus produced an extracellular endo-polygalacturonase (poly (1,4--d-galacturonide) glycanohydrolase EC 3.2.1.15) when grown in a synthetic medium containing one of a variety of pectic substances or galacturonic acid. The highest level of enzyme activity (15.5 VU-ml^–1) was obtained after 72 h of growth on 1.0% low-methoxyl pectin. The enzyme, purified by gel filtration (Sephadex G-100) after repeated ammonium sulphate precipitation and dialysis, showed only one band by polyacrylamide gel electrophoresis and had the following properties: mol wt (MW_r) 41000 dal; isoelectric point (pl) = 8.10 ± 0.10; optimum temperature and pH for activity around 37°C and pH 3.75, respectively; pH stability in the pH range 4.0 to 8.0; complete heat inactivation after 10 min at 55°C; Km and Vmax values 5.7· 10^–1 mg·ml^–1 and 5.1 · 10^–1 mmoles·min^–1, respectively.     

Purification and characterization of a newly screened microbial peptide amidase

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39–45°C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30°C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg²⁺, EDTA, d-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to l-enantiomers in the C-terminal position.     

Purification and some properties of a carboxypeptidase B from dogfish Scyliorhinus canicula

A carboxypeptidase B (CPB) has been purified from dogfish (Scyliorhinus canicula) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o-phenanthroline) and by Hg²⁺. It is activated by Co²⁺, l-cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, K_m and k_cat, of native enzyme, Co²⁺-activated CPB and heat-treated CPB have been determined     

Isolation and properties of carboxypeptidase from leaves of wounded tomato plants

A carboxypeptidase was purified to homogeneity from upper, unwounded leaves of tomato plants in which carboxypeptidase activity had been induced to increase over three-fold by severely wounding the lower leaves. The carboxypeptidase was purified by ammonium sulfate precipitation, affinity chromatography, and finally by gel permeation chromatography. Electrophoresis at pH 4.3 and isoelectric focusing showed only a single band. The isoelectric point was 5.2 and the MW 105 000. Tomato carboxypeptidase possessed both peptidase and esterase activities and it sequentially hydrolysed amino acids from the carboxyl-terminal end of insulin chain B. It was optimally active at pH 6–7 on peptidase substrates, and at pH 8 on esterase substrates. The enzyme was inhibited by diisopropylfluorophosphate and incorporated 1 mol of DFP-[³H]. per mol of enzyme. Both peptidase and esterase activities were strongly inhibited by HgCl₂ but not by p-hydroxymercuribenzoate or iodoacetamide. Carboxypeptidase inhibitor from potatoes did not inhibit the enzyme.     

Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase

Summary The carboxypeptidase previously described³ that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mm MgCl₂ or 30 mm KCl. Mercaptoethanol (50 mm), colchicine (0.2 mm) and tyrosine (0.2 mm) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mm), deoxycholate (0.5%), CuCl₂ (0.1 mm), ZnC1₂ (0.1 mm) and NaCl or KCI (240 mm) had a strong inhibitory effect. The optimal pH of this enzyme. was 6.3–7.A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg²⁺, K^– and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl₂ was 3–20 mm and of KCl was 10–30 mm. For ADP the maximal activity was at 0.3 mm or higher.An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.     

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH – and temperature optima of 3.9 and 50°C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-α-1,4-polygalacturonase (exo-PG I). K_m and V_max for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U × mg⁻¹, respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.     

Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel–nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS–PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH₂-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues −180 to −175, NH₂-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH₂-terminal sequence, LDXNQLY, corresponding to residues −73 to −67 of the proregion peptide and thus were generated by removal of 126 residues from the NH₂-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37°C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k_cat/K_m) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM⁻1 s⁻¹, respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing 2 × 10⁹ High Five cells.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Isolation and characterization of a novel intracellular glucosyltransferase from the acarbose producer <Emphasis Type="Italic">Actinoplanes</Emphasis> sp. CKD485-16

A novel intracellular glucosyltransferase (GTase) was isolated from cells of Actinoplanes sp. CKD485-16—acarbose-producing cells. The enzyme was purified by DEAE-cellulose and G75-40 Sephadex chromatography. The molecular mass of the enzyme was estimated to be 62 kDa by SDS-polyacrylamide gel electrophoresis, and its isoelectric point (pI) was pH 4.3. The N-terminal sequence of the GTase consisted of NH₂-Ser-Val-Pro-Leu-Ser-Leu-Pro-Ala-Glu-Trp. The optimum pH and temperature were 7.5 and 30°C. The enzyme was stable in a pH range of 5.5–9.0 and below 40°C. Enzymatic reactions were performed by incubating the GTase with various substrates. The GTase converted acarbose into component C, maltose into trehalose, and maltooligosaccharides into maltooligosyl trehaloses. The reactions were reversible. Various acarbose analogs were tested as inhibitors against the GTase as a means to suppress component C formation. Valienamine was the most potent, with an IC₅₀ value of 2.4×10^–3 mM and showed a competitive inhibition mode.