Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling

Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 × 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.     

Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily

The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)8 barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP). Of particular interest are PTE, which is thought to have evolved within the last several decades, and PHP, an amidohydrolase superfamily member of unknown function, and the closest known homologue of PTE. We found a diverse and partially overlapping pattern of promiscuous activities in these enzymes, including a significant lactonase activity in PTE, esterase activities in both PTE and PHP, and a weak PTE activity in DHO. Directed evolution was applied to improve the promiscuous esterase activities of PTE and PHP. Remarkably, the most recurrent mutation increasing esterase activity in PTE, or PHP, maps to the same location in their superposed 3D structures. The evolved variants also exhibit newly acquired promiscuous activities that were not selected for, including very weak, yet measurable, paraoxonase activity in PHP. Our results illustrate the mechanistic, structural, and evolutionary links between these enzymes, and highlight the importance of studying laboratory evolution intermediates that might resemble node intermediates along the evolutionary pathways leading to the divergence of enzyme superfamilies.     

The histidine 115-histidine 134 dyad mediates the lactonase activity of mammalian serum paraoxonases

Serum paraoxonases (PONs) are calcium-dependent lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the lactonase mechanism of mammalian PON1, a high density lipoprotein-associated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the lactonase mechanism of PON1 and PON3. The pH-rate profile determined for the lactonase activity of PON1 indicated an apparent pK(a) of approximately 7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His(115), His(134), His(184), His(285), Asp(183), and Asp(269)). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the lactonase activity of PONs. The results indicate that the lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His(115)-His(134) dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad.     

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (k(cat)/K(m)) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.     

Divergence and convergence in enzyme evolution: parallel evolution of paraoxonases from quorum-quenching lactonases

We discuss the basic features of divergent versus convergent evolution and of the common scenario of parallel evolution. The example of quorum-quenching lactonases is subsequently described. Three different quorum-quenching lactonase families are known, and they belong to three different superfamilies. Their key active-site architectures have converged and are strikingly similar. Curiously, a promiscuous organophosphate hydrolase activity is observed in all three families. We describe the structural and mechanistic features that underline this converged promiscuity and how this promiscuity drove the parallel divergence of organophosphate hydrolases within these lactonase families by either natural or laboratory evolution.     

Connectivity between Catalytic Landscapes of the Metallo-β-Lactamase Superfamily

The expansion of functions in an enzyme superfamily is thought to occur through recruitment of latent promiscuous functions within existing enzymes. Thus, the promiscuous activities of enzymes represent connections between different catalytic landscapes and provide an additional layer of evolutionary connectivity between functional families alongside their sequence and structural relationships. Functional connectivity has been observed between individual functional families; however, little is known about how catalytic landscapes are connected throughout a highly diverged superfamily. Here, we describe a superfamily-wide analysis of evolutionary and functional connectivity in the metallo-β-lactamase (MBL) superfamily. We investigated evolutionary connections between functional families and related evolutionary to functional connectivity; 24 enzymes from 15 distinct functional families were challenged against 10 catalytically distinct reactions. We revealed that enzymes of this superfamily are generally promiscuous, as each enzyme catalyzes on average 1.5 reactions in addition to its native one. Catalytic landscapes in the MBL superfamily overlap substantially; each reaction is connected on average to 3.7 other reactions whereas some connections appear to be unrelated to recent evolutionary events and occur between chemically distinct reactions. These findings support the idea that the highly distinct reactions in the MBL superfamily could have evolved from a common ancestor traversing a continuous network via promiscuous enzymes. Several functional connections (e.g., the lactonase/phosphotriesterase and phosphonatase/phosphodiesterase/arylsulfatase reactions) are also observed in structurally and evolutionary distinct superfamilies, suggesting that these catalytic landscapes are substantially connected. Our results show that new enzymatic functions could evolve rapidly from the current diversity of enzymes and range of promiscuous activities.     

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.     

A Common Mutation in Paraoxonase-2 Results in Impaired Lactonase Activity

Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys³¹¹ variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP.     

Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 °C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 °C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.     

Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.     

Structure and specificity of a quorum-quenching lactonase (AiiB) from Agrobacterium tumefaciens

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called "quorum-quenching" enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus

Background

A new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa.

Methodology/Principal Findings

Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins.

Conclusions/Significance

We show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.     

Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k_cat/K_m) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.     

Biochemical basis for hydrolysis of organophosphorus by a marine bacterial prolidase

Extensive application of synthesized organophosphorus compounds (OPs) leads to pollutant accumulation and enhanced eco-toxicity. Hydrolysis of phosphotriester bonds catalyzed by evolved microbial enzymes is a key step for detoxification of OPs. Here, a new marine bacterial prolidase OPAA4301 exhibiting promiscuous phosphotriesterase activity was isolated and systematically characterized. The homo-tetrameric enzyme OPAA4301 can catalyze the hydrolysis of both amido bond and phosphotriester bond. Manganese ions were observed to be essential for its catalytic integrity, and in vitro substitution of manganese ions by different metal cofactors led to decreased activity. We also revealed cooperation pattern of metal ligands and substrate-binding residues on OP hydrolysis by mutational analysis. Metal-binding sites together with Arg418 in the large-binding pocket of the enzyme were found to be indispensable for catalytic ability. Substitution mutation of small- and large-binding pocket residues caused significant variation in phosphotriesterase activity, and leaving group sites appeared to be involved in the catalytic process as substrate affinity regulators. Our study gave an overall biochemical understanding on the organophosphorus hydrolysis pattern of the newly identified marine bacterial prolidase and provided ideas for protein engineering to expand its application in the bioremediation field.     

MomL,a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C₆-HSL is ∼2.9 × 10⁵ s⁻¹ M⁻¹. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.     

Rhodococcus lactonase with organophosphate hydrolase (OPH) activity and His6-tagged OPH with lactonase activity: evolutionary proximity of the enzymes and new possibilities in their application

Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46–50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His₆-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His₆-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His₆-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells.     

The Evolutionary Origins of Detoxifying Enzymes: THE MAMMALIAN SERUM PARAOXONASES (PONs) RELATE TO BACTERIAL HOMOSERINE LACTONASES*

Serum paraoxonases (PONs) are detoxifying lactonases that were first identified in mammals. Three mammalian families are known, PON1, 2, and 3 that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity. Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. Both the mammalian ancestor and the characterized bacterial PONX_OCCAL were found to efficiently hydrolyze N-acyl homoserine lactones that mediate quorum sensing in many bacteria, including pathogenic ones. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like “quorum-quenching” lactonases. The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying lactonase activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.     

Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34-41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%-16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life=7.3 h) than that from SBL11 (half-life=6.4 h at 50 degrees C), while both showed the same temperature optimum of 37 degrees C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.