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The article discusses a study which performs purification and characterization of catechol 2,3-dioxygenase (C23O) from the aniline degradation pathway of Acinetobacter species. The purification of C23O was obtained from cell extract of recombinant Escherichia coli. It is revealed that the wild-type enzyme suffered from substrate inhibition at >2 micrometers, while the mutant enzyme loosened substrate inhibition.

Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6-aminohexanoate-dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. K172. More than 95% of the enzyme activity was lost upon incorporation of 1-1.5 molecules inhibitor/subunit of the enzyme. The tryptic peptide of the labeled enzyme was purified by HPLC (reverse-phase partition) and its amino acid sequence was identified. Radioactivity was found to be incorporated into an 8-amino-acid peptide (108His-Leu-Leu-Met-Ser-Val-SerLys115). Amino acid alteration from Set to Ala at the position 112 by site-directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme). These results indicate that Ser112 is essential for the activity. [ABSTRACT FROM AUTHOR]

6-Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6-aminohexanoic acid cyclic dimer (1,8-diazacyclotetradecane-2,9-dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed. (1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G-200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant (s20) was estimated to be 5.7 S from the sedimentation velocity. (2) The enzyme had an optimum pH of 7.3 when 6-aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45 °C and 50 °C respectively. The optimum temperature for activity was 33 °C. The Michaelis-Menten constant (Km) was 6 mM toward the dimer, and the turnover number was 8 s-1 assuming a molecular weight of 100 000. (3) This enzyme was strongly inhibited by 1 µM diisopropylphosphofluoridate and 10 µM pchloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p-chloromercuribenzoate could be reactivated by 2-mercaptoethanol. (4) This enzyme was only active on 6-aminohexanoic acid cyclic dimer to form 6-aminohexanoyl6-aminohexanoic acid. It was inactive on 6-aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein. (5) The absorption coefficient (A280 nm1%nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer. (6) 6-Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the β-xylosidase (xynB) gene from Bacillus pumilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J. C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539–545]. However, the sequence identified in the present study is quite different from the previously reported one. The total length of the PstI-EcoBI fragment of a plasmid pOXN295 containing the xynB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N-terminal (500 bp) and C-terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified β-xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK-G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40 °C. The Vm value was estimated to be 1.23±0.14 μkat/mg (for p-nitrophenyl β-D-xyloside) and 0.14±0.011 μkat/mg (for xylobiose), while Km was estimated to be 3.9±0.59 mM (for p-nitrophenyl β-D-xyloside) and 8.9±1.19 mM (for xylobiose). [ABSTRACT FROM AUTHOR]

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181 → Asp (EII type) and His266 → Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold. [ABSTRACT FROM AUTHOR]

N-Terminally truncated DNA polymerase from Thermus thermophilus (ΔTth polymerase) lacking 5′-3′ exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, ΔTth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing. The ΔTth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed. Under mutagenic PCR, the mutation rates were 7.0 × 10−4 (Tth) and 8.3 × 10−4 (ΔTth) per nucleotide per cycle of amplification, which were 4–9 times higher than the rates under standard PCR. [ABSTRACT FROM PUBLISHER]

6-Aminohexanoate-cyclic-dimer hydrolase (EI) from Arthrobacter sp. KI72 was expressed in Escherichia coli and purified by anion-exchange chromatography. EI was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as precipitant in imidazole buffer pH 8.0. The crystal is hexagonal, with unit-cell parameters a = b = 130.75, c = 58.23 Å. Diffraction data were collected from native and mercury(II) dichloride-derivative crystals to resolutions of 1.90 and 2.06 Å, respectively. [ABSTRACT FROM AUTHOR]

To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3121 or P3221, with unit-cell parameters a = b = 96.37, c = 113.09 Å. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 Å, respectively. [ABSTRACT FROM AUTHOR]

The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7. [ABSTRACT FROM AUTHOR]

We developed an enzyme-linked immunosorbent assay for an endocrine disrupter, nonylphenol, using a microreactor composed of two reaction vessels stacked vertically through a microfluid filter. The filters constructed by deep X-ray lithography possessed 2100 through-bores (ϕ40×200 μm) in polymethylmethacrylate sheets (ϕ3 mm), which are appropriate for biochemical reactions. Through the optimization of the immunoassay, nonylphenol was quantitatively detected at the range of 0.1–10 ng/ml. [Copyright &y& Elsevier]

Alkylcatechol 2,3-dioxygenase was purified from the cell extract of recombinant Escherichia coli JM109 harboring the alkylcatechol 2,3-dioxygenase gene (bupB) cloned from the butylphenol-degrading bacterium Pseudomonas putida MT4. The purified enzyme (BupB) showed relative meta-cleavage activities for the following catechols: catechol (100%), 4-methylcatechol (572%), 4-n-butylcatechol (185%), 4-n-hexylcatechol (53%), 4-n-heptylcatechol (45%), 4-n-nonylcatechol (10%), 4-tert-butylcatechol (0%), and 3-methylcatechol (33%). The kinetic parameters, namely, K m and V max, for catechol, 4-methylcatechol, and 4-n-butylcatechol, were 23.4, 8.4, and 6.5 μM and 25.8, 76.9, and 18.0 U mg−1, respectively. These results suggest that BupB has broad substrate specificity for 4-n-alkylcatechols. [Copyright &y& Elsevier]

Alkylphenols (APs) are ubiquitous contaminants in aquatic environments and have endocrine disrupting and toxic effects on aquatic organisms. To investigate biodegradation mechanisms of APs, an AP degradation gene cluster was cloned from a butylphenol (BP)-degrading bacterium, Pseudomonas putida MT4. The gene cluster consisted of 13 genes named bupBA1A2A3A4A5A6CEHIFG. From the nucleotide sequences, bupA1A2A3A4A5A6 were predicted to encode a multicomponent phenol hydroxylase (PH), whereas bupBCEHIFG were expected to encode meta-cleavage pathway enzymes. A partial sequence of a putative NtrC-type regulatory gene, bupR, was also found upstream of the gene bupB. This result indicates that APs can be initially oxidized into alkylcatechols (ACs), followed by the meta-cleavage of the aromatic rings. To confirm this pathway, AP degradation tests were carried out using the recombinant P. putida KT2440 harboring the PH genes (bupA1A2A3A4A5A6). The recombinant strain oxidized 4-n-APs with an alkyl chain of up to C7 (≤C7) efficiently and also several BPs including those with an alkyl chain with some degree of branching. Therefore, it was found that PH had a broad substrate specificity for APs with a medium-length alkyl chain (C3–C7). Moreover, the cell extract of a recombinant Escherichia coli harboring bupB (a catechol 2,3-dioxygenase gene) converted 4-n-ACs with an alkyl chain of ≤C9 into yellow meta-cleavage products with a maximum absorbance at 379 nm, indicating that the second step enzyme in this pathway is also responsible for the degradation of ACs with a medium-length alkyl chain. These results suggest that MT4 is a very useful strain in the biodegradation of a wide range of APs with a medium-length alkyl chain, which known nonylphenol-degrading Sphingomonas strains have never degraded. [Copyright &y& Elsevier]

4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP (of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC. [ABSTRACT FROM AUTHOR]

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10 -5  s -1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis., (© 2023 Federation of European Biochemical Societies.)

Biodegradation of synthetic polymers is recognized as a useful way to reduce their environmental load and pollution, loss of natural resources, extensive energy consumption, and generation of greenhouse gases. The potential use of enzymes responsible for the degradation of the targeted polymers is an effective approach which enables the conversion of the used polymers to original monomers and/or other useful compounds. In addition, the enzymes are expected to be applicable in industrial processes such as improving the surface structures of the polymers. Especially, conversion of the solid polymers to soluble oligomers/monomers is a key step for the biodegradation of the polymers. Regarding the hydrolysis of polyamides, three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (NylA), 6-aminohexanoate-dimer hydrolase (NylB), and 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC), are found in several bacterial strains. In this chapter, we describe our approach for the screening of microorganisms which degrade nylons and related compounds; preparation of substrates; assay of hydrolytic activity for soluble and insoluble substrates; and X-ray crystallographic and computational approaches for analysis of structure and catalytic mechanisms of the nylon-degrading enzymes., (© 2021 Elsevier Inc. All rights reserved.)

The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene ( nmoA ) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4'-dihydroxybenzophenone, 4,4'-dihydroxydiphenylether, and 4,4'-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure., Competing Interests: The authors declare no conflict of interest. The funding agency JSPS had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene. These two genes were designated mnpC and mnpB, respectively. The gene products showed high identity with the methylhydroquinone (MHQ) monooxygenase (58%) and the 3-methylcatechol 2,3-dioxygenase (54%) of a different 3M4NP degrader Burkholderia sp. NF100. The transposon mutants converted 3M4NP or MHQ into two identical metabolites, one of which was identified as 2-hydroxy-5-methyl-1,4-benzoquinone (2H5MBQ) by GC/MS analysis. Furthermore, two additional genes (named mnpA1 and mnpA2), almost identical to the p-nitrophenol monooxygenase and the p-benzoquinone reductase genes of Pseudomonas sp. WBC-3, were isolated from the total DNA of strain TSN1. Disruption of mnpA1 resulted in the complete loss of the 3M4NP degradation activity, demonstrating that mnpA1 encodes the initial monooxygenase for 3M4NP degradation. The purified mnpA2 gene product could efficiently reduce methyl p-benzoquinone (MBQ) into MHQ. These results suggest that strain TSN1 degrades 3M4NP via MBQ, MHQ, and 2H5MBQ in combination with mnpA1A2 and mnpCB, existing at different loci on the genome., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a β subunit (9 kDa). Four αβ heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.

Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40-46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 × 10⁶Da.

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme., (Copyright © 2013. Published by Elsevier B.V.)

Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylCp2) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylCp2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å. The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C2221, with unit-cell parameters a=70.84, b=144.90, c=129.05 Å. A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a beta-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K(m) = 15 mM) and turnover (k(cat) = 3.1 s(-1)) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald--COOH and Tyr370, a hydrogen-bonding network from Ser187 to Ald--NH(3) (+), and interaction between Ald--NH(3) (+) and Gln27-O(epsilon) derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with Ald--NH(3) (+) (Kawashima et al., FEBS J 2009; 276:2547-2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a beta-lactamase fold.