P-nitrophenyl Esters Research Articles

Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi.

High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t1/2) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu2+ followed by Ca2+, Ni2+, Co2+, and Mg2+. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. Km, Vmax, and Kcat for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 1011 min-1, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.

Gene cloning, IPTG-independent auto-induction and characterization of a novel hyperstable S9 prolyl oligopeptidase having lipolytic activity from Thermotoga naphthophila RKU-10T with applications

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min−1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0–8.0), and has great stability over a broad range of pH (5.0–10.0), and temperature (40–85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg−1) and olive oil (1182.05 U mg−1). The values of Km (0.576 mM), Vmax (4216 μmol mg−1 min−1), VmaxKm−1 (7319.44 min−1), kcat (1106.74 s−1), and kcatKm−1 (1921.42 mM−1 s−1) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.

Insights into the biodegradation of polycaprolactone through genomic analysis of two plastic-degrading Rhodococcus bacteria.

Polycaprolactone (PCL) is an aliphatic polyester often utilized as a model to investigate the biodegradation potential of bacteria and the involved catabolic enzymes. This study aims to characterize PCL biodegradative metabolic potential and correlate it to genomic traits of two plastic-degrading bacteria-Rhodococcus erythropolis D4 strain, a new isolate from plastic-rich organic waste treatment plant, and Rhodococcus opacus R7, known for its relevant biodegradative potential on polyethylene and similar compounds. After preliminary screening for bacteria capable of hydrolyzing tributyrin and PCL, the biodegradation of PCL was evaluated in R. erythropolis D4 and R. opacus R7 by measuring their growth and the release of PCL catabolism products up to 42 days. After 7 days, an increase of at least one order of magnitude of cell number was observed. GC-MS analyses of 28-day culture supernatants showed an increase in carboxylic acids in both Rhodococcus cultures. Furthermore, hydrolytic activity (~5 U mg-1) on short/medium-chain p-nitrophenyl esters was detected in their supernatant. Finally, a comparative genome analysis was performed between two Rhodococcus strains. A comparison with genes annotated in reference strains revealed hundreds of gene products putatively related to polyester biodegradation. Based on additional predictive analysis of gene products, gene expression was performed on a smaller group of genes, revealing that exposure to PCL elicits the greatest increase in transcription for a single gene in strain R7 and two genes, including that encoding a putative lipase, in strain D4. This work exhibits a multifaceted experimental approach to exploit the broad potential of Rhodococcus strains in the field of plastic biodegradation.

Genome-wide analysis of lipolytic enzymes and characterization of a high-tolerant carboxylesterase from Sorangium cellulosum.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14-37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0-9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited β-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Cloning and characterization of an acidic lipase from a lipolytic bacterium in tempeh

BackgroundLipases have emerged as essential biocatalysts, having the ability to contribute to a wide range of industrial applications. Microbial lipases have garnered significant industrial attention due to their stability, selectivity, and broad substrate specificity. In the previous study, a unique lipolytic bacterium (Micrococcus luteus EMP48-D) was isolated from tempeh. It turns out the bacteria produce an acidic lipase, which is important in biodiesel production. Our main objectives were to clone the acidic lipase and investigate its potential in biodiesel production. ResultIn this study, the gene encoding a lipase from M. luteus EMP48-D was cloned and expressed heterologously in Escherichia coli. To our knowledge, this is the first attempt at the cloning and expression of the lipase gene from Micrococcus luteus. The amino acid sequence was deduced from the nucleotide sequence (1356 bp) corresponded to a protein of 451 amino acid residues with a molecular weight of about 40 kDa. The presence of a signal peptide suggested that the protein was extracellular. A sequence analysis revealed that the protein had a lipase-specific Gly-X-Ser-X-Gly motif. The enzyme was identified as an acidic lipase with a pH preference of 5.0. Fatty acid preferences for enzyme activities were C8 and C12 (p-nitrophenyl esters), with optimum temperatures at 30–40 °C and still remaining active at 80°C. The enzyme was also shown to convert up to 70% of the substrate into fatty acid methyl ester. ConclusionThe enzyme was a novel acidic lipase that demonstrated both hydrolytic and transesterification reactions. It appeared particularly promising for the synthesis of biodiesel as this enzyme’s catalytic reaction was optimum at low temperatures and was still active at high temperatures.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Identification of Cutinolytic Esterase from Microplastic-Associated Microbiota Using Functional Metagenomics and Its Plastic Degrading Potential.

Plastic pollution has threatened biodiversity and human health by shrinking habitats, reducing food quality, and limiting the activities of organisms. Therefore, global interest in discovering novel enzymes capable of degrading plastics has increased considerably. Within this context, the functional metagenomic approach, which allows for unlocking the functional potential of uncultivable microbial biodiversity, was used to discover a plastic-degrading enzyme. First, metagenomic libraries derived from microplastic-associated microbiota were screened for esterases capable of degrading both tributyrin and polycaprolactone. Clone KAD01 produced esterase highly active against p-nitrophenyl esters (C2-C16). The gene corresponding to the enzyme activity showed moderate identity (≤ 55.94%) to any known esterases/cutinases. The gene was extracellularly expressed with a 6× histidine tag in E. coli BL21(DE3), extracellularly. Titer of the enzyme (CEstKAD01) was raised from 21.32 to 35.17U/mL by the statistical optimization of expression conditions and media components. CEstKAD01 was most active at pH 7.0 and 30°C. It was noteworthy stable over a wide pH (6.0-10.0) and temperature (20-50°C). The enzyme was active and stable in elevated NaCl concentrations up to 12% (w/v). Pre-incubation of CEstKAD01 with Mg2+, Mn2+, and Ca2+ increased the enzyme activity. CEstKAD01 displayed an excellent tolerance against various chemicals and solvents. It was determined that 1mg of the enzyme caused the release of 5.39 ± 0.18mM fatty acids from 1g apple cutin in 120min. Km and Vmax values of CEstKAD01 against p-nitrophenyl butyrate were calculated to be 1.48mM and 20.37µmol/min, respectively. The enzyme caused 6.94 ± 0.55, 8.71 ± 0.56, 7.47 ± 0.47, and 9.22 ± 0.18% of weight loss in polystyrene, high-density polyethylene, low-density polyethylene, and polyvinyl chloride after 30-day incubation. The scanning electron microscopy (SEM) analysis indicated the formation of holes and pits on the plastic surfaces supporting the degradation. In addition, the change in chemical structure in plastics treated with the enzyme was determined by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Finally, the degradation products were found to have no genotoxic potential. To our knowledge, no cutinolytic esterase with the potential to degrade polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyvinyl chloride (PVC) has been identified from metagenomes derived from microplastic-associated microbiota.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Front Cover: Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues (Chem. Eur. J. 42/2023)

Vinyl and p-nitrophenyl esters have emerged as alternative substrates for the LovD9 acylation mechanism, showing product yields as high as those obtained by α-dimethylbutyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved. More information can be found in the Research Article by G. Jiménez-Osés, F. López-Gallego and co-workers (DOI: 10.1002/chem.202300911).

Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues.

This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.

Artificial esterase for cooperative catalysis of ester hydrolysis at pH 7

Ester is one of the most prevalent functional groups in natural and man-made products. Natural esterases hydrolyze nonactivated alkyl esters readily but artificial esterases generally use highly activated p-nitrophenyl esters as substrates. We report synthetic esterases constructed through molecular imprinting in cross-linked micelles. The water-soluble nanoparticle catalysts contain a thiouronium cation to mimic the oxyanion hole and a nearby base to assist the hydrolysis. Whereas this catalytic motif readily affords large rate acceleration for the hydrolysis of p-nitrophenyl hexanoate, nonactivated cyclopentyl hexanoate demands catalytic groups that can generate a strong nucleophile (hydroxide) in the active site. The hydroxide is stabilized by the protonated base when the external solution is at pH 7, enabling the hydrolysis of activated and nonactivated esters under neutral conditions.

Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Altering the Regioselectivity of T1 Lipase from Geobacillus zalihae toward sn-3 Acylglycerol Using a Rational Design Approach

The regioselectivity characteristic of lipases facilitate a wide range of novel molecule unit constructions and fat modifications. Lipases can be categorized as sn-1,3, sn-2, and random regiospecific. Geobacillus zalihae T1 lipase catalyzes the hydrolysis of the sn-1,3 acylglycerol chain. The T1 lipase structural analysis shows that the oxyanion hole F16 and its lid domain undergo structural rearrangement upon activation. Site-directed mutagenesis was performed by substituting the lid domain residues (F180G and F181S) and the oxyanion hole residue (F16W) in order to study their effects on the structural changes and regioselectivity. The novel lipase mutant 3M switches the regioselectivity from sn-1,3 to only sn-3. The mutant 3M shifts the optimum pH to 10, alters selectivity toward p-nitrophenyl ester selectivity to C14-C18, and maintains a similar catalytic efficiency of 518.4 × 10−6 (s−1/mM). The secondary structure of 3M lipase comprises 15.8% and 26.3% of the α-helix and β-sheet, respectively, with a predicted melting temperature (Tm) value of 67.8 °C. The in silico analysis was conducted to reveal the structural changes caused by the F180G/F181S/F16W mutations in blocking the binding of the sn-1 acylglycerol chain and orientating the substrate to bond to the sn-3 acylglycerol, which resulted in switching the T1 lipase regioselectivity.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Catalysis and inhibition of ester hydrolysis by encapsulation in micelles derived from designer surfactant TPGS-750-M

The designer surfactant TPGS-750-M is an important technology for green and sustainable chemical synthesis that facilitates a wide variety of organic transformations in water. Despite its growing importance in organic synthesis, many basic aspects of binding and reactivity within TPGS-750-M micelles remain poorly understood. In this work, the impact of TPGS-750-M on the rate of p-nitrophenyl ester hydrolysis kinetics was investigated to probe binding strength within TPGS-750-M micelles and the chemical environment of the micelle interior. We found that the hydrolysis rates of p-nitrophenyl acetate and p-nitrophenyl hexanoate were slowed by the presence of TPGS-750-M by up to 92%, consistent with a hydrophobic encapsulation environment within the micelle interior. The hydrolysis rates of p-nitrophenyl decanoate and p-nitrophenyl hydroxycinnamate were accelerated up to 74-fold by the presence of TPGS-750-M, but only at low surfactant concentrations. We propose that these rate enhancements are due to mixed micelle formation between ester and TPGS-750-M, which positions the reactive ester group within the hydrophilic region of the micelle. This study suggests that there are two distinct chemical environments present in the interior of micelles derived from TPGS-750-M, and that it is possible for very hydrophobic substrates to interact with the polar, hydrophilic region of the micelle. This work also demonstrates that increased hydrophobicity leads to stronger substrate binding within TPGS-750-M micelles.

An artificial self-assembling peptide with carboxylesterase activity and substrate specificity restricted to short-chain acid p-nitrophenyl esters.

Natural enzymes possess remarkable catalytic activity and high substrate specificity. Many efforts have been dedicated to construct artificial enzymes with high catalytic activity. However, how to mimic the exquisite substrate specificity of a natural enzyme remains challenging because of the complexity of the enzyme structure. Here, we report artificial carboxylesterases that are specific for short chain fatty acids and were constructed via peptide self-assembly. These artificial systems have esterase-like activity rather than lipase-like activity towards p-nitrophenyl esters. The designer peptides self-assembled into nanofibers with strong β-sheet character. The extending histidine units and the hydrophobic edge of the fibrillar structure collectively form the active center of the artificial esterase. These artificial esterases show substrate specificity for short-chain acids esters. Moreover, 1-isopropoxy-4-nitrobenzene could function as a competitive inhibitor of hydrolysis of p-nitrophenyl acetate for an artificial esterase.

Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2–C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

Analysis of double-emulsion droplets with ESI mass spectrometry for monitoring lipase-catalyzed ester hydrolysis at nanoliter scale

Microfluidic double-emulsion droplets allow the realization and study of biphasic chemical processes such as chemical reactions or extractions on the nanoliter scale. Double emulsions of the rare type (o1/w/o2) are used here to realize a lipase-catalyzed reaction in the non-polar phase. The surrounding aqueous phase induces the transfer of the hydrophilic product from the core oil phase, allowing on-the-fly MS analysis in single double droplets. A microfluidic two-step emulsification process is developed to generate the (o1/w/o2) double-emulsion droplets. In this first example of microfluidic double-emulsion MS coupling, we show in proof-of-concept experiments that the chemical composition of the water layer can be read online using ESI–MS. Double-emulsion droplets were further employed as two-phase micro-reactors for the hydrolysis of the lipophilic ester p-nitrophenyl palmitate catalyzed by the Candida antarctica lipase B (CalB). Finally, the formation of the hydrophilic reaction product p-nitrophenol within the double-emulsion droplet micro-reactors is verified by subjecting the double-emulsion droplets to online ESI–MS analysis.Graphical abstract

Construction of Zn-heptapeptide bionanozymes with intrinsic hydrolase-like activity for degradation of di(2-ethylhexyl) phthalate

Nanozyme with intrinsic enzyme-like activity has emerged as favorite artificial catalyst during recent years. However, current nanozymes are mainly limited to inorganic-derived nanomaterials, while biomolecule-sourced nanozyme (bionanozyme) are rarely reported. Herein, inspired by the basic structure of natural hydrolase family, we constructed 3 oligopeptide-based bionanozymes with intrinsic hydrolase-like activity by implementing zinc induced self-assembly of histidine-rich heptapeptides. Under mild condition, divalent zinc (Zn2+) impelled the spontaneous assembly of short peptides (i.e. Ac-IHIHIQI-CONH2, Ac-IHIHIYI-CONH2, and Ac-IHVHLQI-CONH2), forming hydrolase-mimicking bionanozymes with β-sheet secondary conformation and nanofibrous architecture. As expected, the resultant bionanozymes were able to hydrolyze a serious of p-nitrophenyl esters, including not only the simple substrate with short side-chain (p-NPA), but also more complicated ones (p-NPB, p-NPH, p-NPO, and p-NPS). Moreover, the self-assembled Zn-heptapeptide bionanozymes were also proven to be capable of degrading di(2-ethylhexyl) phthalate (DEHP), a typical plasticizer, showing great potential for environmental remediation. Based on this study, we aim to provide theoretical references and exemplify a specific case for directing the construction and application of bionanozyme.

PTCL1-EstA from Paenarthrobacter aurescens TC1, a Candidate for Industrial Application Belonging to the VIII Esterase Family

The esterase PTCL1-EstA from Paenarthrobacter aurescens TC1 was expressed in Escherichia coli and characterized. An 1152 bp open reading frame encoding a 383 amino acid polypeptide was successfully expressed, the C-terminally His6-tagged PTCL1-EstA enzyme was purified, and the predicted molecular mass of the purified PTCL1-EstA was 40.6 kDa. The EstA family serine hydrolase PTCL1-EstA belongs to the esterase family VIII, contains esterase-labeled S-C-S-K sequences, and homologous class C beta-lactamase sequences. PTCL1-EstA favored p-nitrophenyl esters with C2-C6 chain lengths, but it was also able to hydrolyze long-chain p-nitrophenyl esters. Homology modelling and substrate docking predicted that Ser59 was an active site residue in PTCL1-EstA, as well as Tyr148, Ala325, and Asp323, which are critical in catalyzing the enzymatic reaction of p-nitrophenyl esters. PTCL1-EstA reached the highest specific activity against p-nitrophenyl butyrate (C4) at pH 7.0 and 45 °C but revealed better thermal stability at 40 °C and maintained high relative enzymatic activity and stability at pH 5.0–9.0. Fermentation medium optimization for PTCL1-EstA increased the enzyme activity to 510.76 U/mL, tapping the potential of PTCL1-EstA for industrial production.