Hydrolytic Activity Research Articles

Antifungal, Antibacterial, and Antioxidant Activities of Camel Whey Protein Hydrolysates

Antifungal, Antibacterial, and Antioxidant Activities of Camel Whey Protein Hydrolysates

Immunoglobulin constant regions provide stabilization to the paratope and enforce epitope specificity.

Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharide from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extending to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results and provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.

Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum

ABSTRACT Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.

Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

The individual contributions of blaB, blaGOB and blaCME on MICs of β-lactams in Elizabethkingia anophelis.

The blaB, blaGOB and blaCME genes are thought to confer β-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. To determine the individual contributions of β-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. Scarless gene deletion of one or more β-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of β-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 μM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (−)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (− 80, − 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including − 80, − 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

Characterization of glucose/non-glucose-tolerant β-glucosidases from the metatranscriptome in compost

Functional microbial communities producing β-glucosidases differentially regulate the expression of glucose/non-glucose-tolerant β-glucosidases in response to carbon catabolite repression (CCR), which is a common phenomenon. To better understand their biological roles in a composting environment, four representative β-glucosidase genes were cloned from the metatranscriptome of the compost for expression and characterization. BGLA contains conserved sites Trp168 and Leu173, associated with glucose tolerance. In the presence of 100 mM glucose, BGLA's hydrolysis activity increases by 80%. BGLB also contains Trp168 and Leu173; however, its hydrolysis activity is significantly inhibited by glucose. On the contrary, Trp168 of BGLC was replaced by Phe, yet its hydrolysis activity increased by 20% in the presence of 25 mM glucose. However, BGLD did not exhibit any enzymatic activity. Molecular docking and dynamic simulations revealed the structural basis of glucose tolerance. Metatranscriptomic analysis of the four genes showed that functional microbial communities upregulated or downregulated gene expression in response to different CCR environments to maintain overall carbon metabolic balance. These findings demonstrate that the enzymatic activity characteristics of individual glucose/non-glucose-tolerant β-glucosidases are consistent with their transcriptional regulation under CCR in the complex composting environment.

Effect of grain structure and cooking method on starch digestibility and antioxidant activity of amaranth (Amaranthus caudatus L.) grains during simulated gastrointestinal digestion

Amaranth has received widespread interest as a high-quality source of nutrition. This study examined changes in starch hydrolysis and antioxidant activity of intact or slurry of amaranth grains, which were cooked with different methods, such as boiled and roasted, during simulated in vitro gastrointestinal digestion. The starch digestibility of amaranth samples was analyzed using starch hydrolysis percentage and digestion kinetics during simulated digestion. Simultaneously, TPC and antioxidant assays, such as ABTS, FRAP, and MIC, assessed the changes in antioxidant potential during digestion. The results showed that the slurry increased starch hydrolysis and antioxidant properties compared with intact grain. Boiling and roasting also showed increased antioxidant activity and starch hydrolysis during digestion. Whether intact grains or slurries, the roasted samples had lower starch hydrolysis and higher antioxidant properties during digestion than the boiled samples. The equilibrium starch hydrolysis percentages of the roasted and boiled intact grains were 15.50 and 67.41 %, and those TPC were 662.78 and 470.21 mg GAE/100 g sample (db.), respectively. In this study, roasting was more beneficial in delaying starch hydrolysis and improving antioxidant activity than boiling. In addition, maintaining the grain state was more conducive to controlling starch hydrolysis. Our findings could be beneficial for the amaranth consumers.

Characterization of Fish Skin Hydrolysates Exhibiting Dipeptidyl Peptidase IV Inhibitory Activity

Dipeptidyl peptidase IV (DPP-IV) inhibitors are antidiabetic drugs that can lower blood sugar levels. There are still few reports on the DPP-IV inhibitory activity of peptides obtained from discarded fish skin. Therefore, we prepared various enzymatic hydrolysates using the skins of six fish species and investigated their DPP-IV inhibitory effects. As a result, it was found that the DPP-IV inhibitory activity of yellowtail hydrolysate by Alcalase was higher than that of other enzymes. In addition, the IC50 after ethanol fractionation was found to be lower in yellowtail and eel skin hydrolysate. Amino acid composition analysis showed that the hydrolysate obtained from the skin of the yellowtail contained the highest amount of Gly, followed by Pro, Hyp, and Ala, indicating that it was a peptide derived from type I collagen. Fractionation with ethanol showed that the DPP-IV inhibitory components were contained in the low molecular weight fraction. The artificial digestion test observed no DPP-IV inhibitory activity or average molecular weight change. The DPP-IV inhibitory peptide obtained from fish skin has the potential to be applied as a food material to various food products.

Catalytic promiscuity, cytotoxicity and protein cleavage mediated by mononuclear copper(II) complexes: oxidative and hydrolytic activities

In this work, we describe the synthesis and structural and spectroscopic characterizations of two copper(II) complexes, [(Cu)(L1)(Cl2)].4H2O (1), where L1 is 2-(pyridin-2-yl)-1,3-bis(pyridin-2-ylmethyl)-hexahydropyrimidine, and [Cu(L2)](ClO4)2 (2), where L2 is N1,N1,N3-tris(pyridin-2-ylmethyl)propane-1,3-diamine. The crystalline structure analysis indicates that both complexes are mononuclear and pentacoordinated compounds. In the case of complex 1, it bears a neutral charge, with the ligand coordinating to the Cu(II) center in a tridentate manner, utilizing N-donor atoms. Additionally, two cis-chlorido ligands completing the coordination sphere. On the other hand, complex 2 is a cationic complex, with the ligand coordinated in a pentadentate manner, with only N-donor atoms for coordination around the Cu(II). Complex 1 showed catalytic promiscuity, displaying hydrolytic and oxidative activity. The hydrolytic activity was investigated using bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) as a phosphodiester substrate and applying the Michaelis–Menten approach, obtaining the following kinetic parameters: kcat = 1.21 × 10-4 s−1; KM = 3.35 × 10-3 molL-1. Complex 1 also accelerates the oxidation of the 3,5-di-tert-butylcatechol (3,5-DTBC) substrate with catalytic efficiency equal to 6.11 L mol-1s-1. Interestingly, we demonstrated that this Cu(II) complex 1 can also cleave the very stable peptide bonds from BSA (bovine serum albumin) through an oxidative mechanism using ascorbate as a reducing agent. Complexes 1 and 2 show good cytotoxic activity against two cancer cell lines: A431 from human squamous cell carcinoma and K562 from chronic myeloid leukemia. This study showed that complexes 1 and 2 are selective towards cancer cells.

New insights on the analysis of the causes of glossy lustre on the surface of ancient Chinese bronze mirrors

Ancient Chinese bronzes, particularly bronze mirrors, often develop a fatty, oily, or greasy film on their surface, known as patina. This film can appear silvery, green, or black. Bronze mirror samples from the Spring and Autumn Period (770–476/403 BCE) to the Ming Dynasty (1368–1644 CE) were analyzed using a systematic approach that included super depth of field three-dimensional microscopy, scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM–EDS), Raman spectroscopy, and THM-Py-GC/MS. The analysis indicates the presence of advanced organic acid lead in the matrix of ancient bronze mirrors. The ancient existence of the lost wax method combined with the mould method of bronze casting technology is verified through simulation experiments and ancient literature. Ancient bronze mirrors may have utilized this technique, with advanced organic acid lead remaining in the mirror mould that entered the bronze mirror matrix during pouring. The Lead organic acids in bronze mirrors readily precipitate on the surface and cover the mirror. The change in the greasy sheen on the surface of the bronze mirror may be related to the degree of corrosion. If the mirror's corrosion level is low, the organic acid lead film remains transparent. If the level of corrosion is significant, the organic acid lead film may darken in color due to oxidation, aging, hydrolysis, or microbial activity.

Emulsifying property, antioxidant activity, and bitterness of soybean protein isolate hydrolysate obtained by Corolase PP under high hydrostatic pressure

Enzymatic hydrolysis of proteins can enhance their emulsifying properties and antioxidant activities. However, the problem related to the hydrolysis of proteins was the generation of the bitter taste. Recently, high hydrostatic pressure (HHP) treatment has attracted much interest and has been used in several studies on protein modification. Hence, the study aimed to investigate the effects of enzymatic hydrolysis by Corolase PP under different pressure treatments (0.1, 100, 200, and 300 MPa for 1–5 h at 50 °C) on the emulsifying property, antioxidant activity, and bitterness of soybean protein isolate hydrolysate (SPIH). As observed, the hydrolysate obtained at 200 MPa for 4 h had the highest emulsifying activity index (47.49 m2/g) and emulsifying stability index (92.98 %), and it had higher antioxidant activities (44.77 % DPPH free radical scavenging activity, 31.12 % superoxide anion radical scavenging activity, and 61.50 % copper ion chelating activity). At the same time, the enhancement of emulsion stability was related to the increase of zeta potential and the decrease of mean particle size. In addition, the hydrolysate obtained at 200 MPa for 4 h had a lower bitterness value and showed better palatability. This study has a broad application prospect in developing food ingredients and healthy foods.

Integrating metal-organic framework particles on fabric membranes for decontaminating toxic organophosphates

Zirconium-based metal-organic frameworks (Zr-MOFs) with periodic Lewis acidic nodes have demonstrated impressive catalytic activity in the hydrolysis of organophosphorus nerve agents. Nevertheless, the powdered form of Zr-MOFs and the necessity of a base-buffered aqueous solution during the catalytic reaction pose significant challenges to their practical utilization. In this study, we demonstrate the efficient hydrolysis of an organophosphorus nerve agent simulant, dimethyl-4-nitrophenyl phosphate (DMNP), in both pure water and the solid phase under high humidity conditions, catalyzed by a membrane material. This material, denoted as Im@MOF-808/PVDF, was synthesized through the integration of MOF-808 particles onto PVDF membrane fibers, with imidazole (Im) molecular bases incorporated into the pores of MOF-808. Our findings emphasize the excellent flexibility and processability inherent in Im@MOF-808/PVDF. More notably, it exhibits exceptional catalytic performance in the hydrolysis of DMNP in pure water. Additionally, it demonstrates fair catalytic activity for solid-phase DMNP hydrolysis under high humidity conditions. These features position Im@MOF-808/PVDF as one of the most promising protective materials, showcasing substantial practical applicability.

Performance and microbial community composition of full-scale high-rate cascade sludge digestion system via pie-shaped reactor configuration

A full-scale high-rate cascade anaerobic digestion (CAD) system was evaluated for its ability to enhance enzymatic sludge hydrolysis. The system included a newly built digester, innovatively divided into three pie-shaped compartments (500 m3 each), followed by an existing, larger digester (1500 m3). The system treated a mixture of waste activated sludge and primary sludge, achieving a stable total chemical oxygen demand reduction efficiency (56.1 ± 6.8 %), and enhanced sludge hydrolytic enzyme activities at a 14.5-day total solids retention time (SRT). High-throughput sequencing data revealed a consistent microbial community across reactors, dominated by consortia that govern hydrolysis and acidogenesis. Despite relatively short SRTs in the initial reactors of the CAD system, acetoclastic methanogens belonging to Methanosaeta became the most abundant archaea. ‬‬‬‬‬‬‬‬‬‬‬‬‬ This study proves that the CAD system achieves stable sludge reduction, accelerates enzymatic hydrolysis at full-scale, and paves the way for its industrialization in municipal waste sewage sludge treatment.

Exploration of novel trehalases from cold-adapted Variovorax sp. PAMC28711: Functional characterization

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) μmolmg−1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) μmolmg−1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.

Efficacious bioconversion of alginate/cellulose to value-added oligosaccharides by alginate-degrading GH5 endoglucanase from Trichoderma asperellum

Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 β-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (∼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0–5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.

Some selected properties of the recombinant aminoacylase from Escherichia coli LGE 36

Background: Aminoacylase has seen extensive use in the synthesis of L-amino acids. L-amino acids are widely used in the food and medical industries, as well as in healthcare. The effect of metal ions and inhibitors on the enzyme activity of recombinant intracellular aminoacylase of Escherichia coli LGE 36 was studied. Acetyl-D,L-methionine and glycyl-L-methionine dipeptide were used as substrates. It has been shown that with the addition of Co2+ ions, the acylase activity of the recombinant enzyme towards N-acetyl-D,L-methionine increases 3 times. In the case of glycyl-L-methionine, the peptidase activity of the recombinant aminoacylase of Escherichia coli LGE 36 increases more than 40 times. A number of other metal ions studied do not stimulate the hydrolytic activity of the enzyme, moreover some of them even inhibit it. PCMB completely inactivates the recombinant aminoacylase, EDTA reduces enzyme activity by 85%. Objective: This study aims to investigate how metal ions and inhibitors affect the enzyme activity of recombinant intracellular aminoacylase Escherichia coli LGE 36. Methods: In this study, a recombinant strain-producer of aminoacylase derived from Escherichia coli LGE 36 was employed. Escherichia coli cells were cultured in M9 minimal medium supplemented at 37°C. To assess aminoacylase activity, assays were conducted at 37°C using a reaction mixture of 0.2 ml final volume comprising 100 mM Na, K-phosphate buffer at pH 7.0, 0.2 mM CoCI2, 40 mM N-acetyl-D, L-methionine, and the enzyme in the appropriate concentration. Acylase activity was quantified as the amount of enzyme catalyzing the formation of 1µmol of L-methionine within 1 minute at 37°C and pH 7.0. Results: Some characteristics of the recombinant intracellular aminoacylase from E. coli LGE 36 were determined. For investigating the impact of metallic ions and inhibitors, acetylmethionine and glycylmethionine were employed as the substrates. Conclusion: The present study and the choice of right strategies for functional food products allows optimizing the process for production of optically active amino acids using the recombinant enzyme to obtain food and feed additives. Keywords: ion, metals, inhibitors, acetylmethionine, glycylmethionine, recombinant aminoacylase from Escherichia coli, enzyme.

A Novel Thermo-Alkaline Stable GDSL/SGNH Esterase with Broad Substrate Specificity from a Deep-SeaPseudomonas sp.

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60°C, respectively, but it retained more than 50% of its activity over the pH range of 7-11 and temperature range of 10-75°C. In addition, EstGoM was tolerant of up to 1M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and β-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications.