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Abstract Background Laccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods. Results This study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method. Conclusions This study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications. Graphical Abstract

Background: Laccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods. Results: This study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method. Conclusions: This study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications. [ABSTRACT FROM AUTHOR]

Previously, elastase activities have been found in some microorganisms and presented by metalloproteases, which may play an important role in helping bacterium invasiveness and establishment of infection. The elastases from Bacillus species are serine proteases, however, to date there are few reports on systematic study of these enzymes producers among representatives of Bacillus, as well as studies of their properties. The aim of this work was to study some physicochemical characteristics of partially purified elastase enzyme from a number of Bacillus sp. strains. Methods. The objects of the study were strains of Bacillus sp. (L1, L2, L9) isolated from the dry grass of the coastal zone of the Kinburn split (Mykolaiv region). Cultures were grown under submerged cultivation at 28°C, with a shaking speed of the nutrient medium of 230 rpm for 2 days. Methods of determining elastase activity in the culture liquid supernatant were used. Results. It has been shown that Bacillus sp. L9, L1, and L2 are characterized by high levels of elastase activity (35.80, 28.0, and 33.80 U/mL, respectively). The maximum synthesis of elastase occurs on the second day of cultivation of the producer at 28 °C and shaking at 230 rpm. Maximum hydrolysis of elastin by Bacillus sp. L1 and L2 are carried out at 40 °C but at different pH optima. Thus, for the preparation of Bacillus sp. L1, optimal pH is 8, and for L2 - 10. For the complex enzyme preparation of Bacillus sp. L9, maximum hydrolysis of elastin occurs at pH 9 and temperature 50 °C. [ABSTRACT FROM AUTHOR]

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen.

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans' producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity. [ABSTRACT FROM AUTHOR]

Eco-enzymes are one of the potential sources for obtaining amylolytic bacterial isolates. The study aims to screen amylolytic bacteria from eco-enzymes, characterize semi-purification amylase, and identify amylolytic bacteria molecularly using the 16S rRNA gene. Screening for amylolytic bacteria was carried out by measuring the amylolytic index on a Nutrient agar medium containing 1% tapioca starch. The amylolytic isolate which had the highest index and was non-pathogenic was selected for the amylase characterization process. Testing of amylase activity was carried out using the Bernfeld method while the protein enzymes were measured using the Bradford method. The extracellular extract was concentrated using ammonium sulfate precipitation. PKL2 is gram-positive bacteria that was derived from eco-enzymes with the highest amylolytic indexes of 1.77, which were not pathogenic on the blood agar test. Optimum amylase was produced by PKL2 at the stationary phase at 21 h. The optimum pH and temperature of the amylase activity were observed to be 7.0 and 50°C, respectively. The amylase enzyme from PKL2 increased its purity 1.82-fold upon precipitation of ammonium sulfate at a concentration of 60%. Identification of bacteria based on molecular identification showed that PKL2 obtained was putatively identified as Bacillus amyloliquefaciens. [ABSTRACT FROM AUTHOR]

ABSTRACT: β-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (pKa) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase.

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme. [ABSTRACT FROM AUTHOR]

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5–5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (Pul PY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of Pul PY2 -Q13H and Pul PY2 -I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant Pul PY2 -Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to Pul PY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface. [ABSTRACT FROM AUTHOR]

The surge in vancomycin-resistant enterococci (VRE) strains poses a serious threat to public and clinical health. VRE strains are the leading cause of multi-drug resistant enterococcal infections and are commonly acquired from medical devices. Therefore, it is essential to discover new antibacterial targets and drugs for this pathogen. Alanine racemase could be a valuable drug target due to its crucial role in E. faecium survival. Alr from E. faecium (EF_Alr) was heterologously produced and purified from E. coli., and the steady-state kinetic constants were determined at different pH values. Using a coupled reaction with L-alanine dehydrogenase, rate of production of NADH was measured at 340 nm to observe EF_Alr activity in the D- to L-alanine direction. The highest catalytic efficiency, 8.61 ± 0.5 s-1 mM-1, was found at pH 9. Additionally, the tentative active site residues, Lys40 and Tyr268, for the alanine racemization reaction were assigned by homology modeling and sequence comparison studies. Using UCSF Chimera, the structure of the EF_Alr homology model was superimposed and compared to the crystal structure of Alr from E. faecalis. [ABSTRACT FROM AUTHOR]

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME. [ABSTRACT FROM AUTHOR]

The review discusses the basic biochemical properties of the known recombinant chymosins that are currently used in cheesemaking or may potentially be used as industrial milk coagulants. The parameters of the Michaelis–Menten kinetics, milk-clotting activity, proteolytic activity, specificity, and the dependence of coagulation ability on pH and calcium-ion concentration are considered. [ABSTRACT FROM AUTHOR]

Abstract Background Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. Results In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. Conclusions We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.

The use of a modified, four-stage purification scheme allowed us to obtain electrophoretically homogeneous preparations of constitutive forms of succinate dehydrogenase from maize (Zea mays L.) scutellum in the late stages of seed germination. It has been established that the isoenzymes differed significantly in their quaternary structure. Thus, succinate dehydrogenase 1 turned out to be a heterotetramer, and succinate dehydrogenase 2 turned out to be a heteroterodimer. It was found that their main catalytic and kinetic characteristics also differed, in particular, by their affinity to the substrate (succinate) and the value of pH optimum of the catalyzed reaction. Succinate dehydrogenase 2 was characterized by enhanced resistance to a specific inhibitor—malonate. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Hydrogen-bond-based approach was conducted to improve acidic adaptation of pullulanase. • The pH optimum of the B. acidopullulyticus pullulanase mutant shifted from 5.0 to 4.0. • The mutant exhibited increased tolerance against acid-mediated denaturation. • Kinetic parameters and p K a prediction confirmed the improved efficacy of the mutant. • The maximum d -glucose content for the mutant reached 97.4% after incubation for 40 h. Abstract Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (Ba Pul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (p K a) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of Ba Pul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and p K a prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d -glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced energy consumption. [ABSTRACT FROM AUTHOR]

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS. [ABSTRACT FROM AUTHOR]

Background: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. Results: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. Conclusions: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis. [ABSTRACT FROM AUTHOR]

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The ability to form biofilms in natural isolate Bacillus subtilis 168 and mutants with deleted genes of regulatory proteins AbrB, DegU, CcpA, and SpoOA, constructed on its basis, was investigated to elucidate the pathways regulating biofilm formation in B. subtilis. The B. subtilis 168 wild-type forms a biofilms in the liquid medium with maximum at 48th hour of culture growth. pH optimum for the biofilm formation in the wild-type strain is in the range of 7.4-8.0. Temperature optimum was in the range of 22 to 45 °C. The level of biofilm formation for all regulatory mutants was lower than that in the wild-type for 40-50 %. Temperature and pH optima for the mutant strains are the same as for the wild-type strain-7.4-8 pH and temperature of 22-45 °C. [ABSTRACT FROM AUTHOR]

The enzymatic decarboxylation of L-lysine using lysine decarboxylase is a promising biological approach for producing cadaverine, a versatile platform chemical for bio-polyamides. However, due to the problem with elevated pH in the reaction solution during the enzymatic process, it is desirable to use lysine decarboxylases effectively active in alkaline conditions. In this study, the catalytic properties of three lysine decarboxylases from Selenomonas ruminantium (srLDC), Vibrio vulnificus (vvLDC), and Geobacillus thermodenitrificans (gtLDC) were characterized, and the applicability of the enzymes in alkaline conditions was investigated. Among the three enzymes, only vvLDC exhibited effective activity in alkaline pH conditions. The conversion rate of vvLDC was 1.5-fold higher than that of srLDC and 5.3-fold higher than that of gtLDC in pH 9.0. The results indicate that vvLDC is more advantageous than srLDC and gtLDC for the enzymatic conversion of L-lysine to cadaverine in alkaline conditions. [ABSTRACT FROM AUTHOR]

Two α-galactosidases, AgaAJB07 from Mesorhizobium and AgaAHJG4 from Streptomyces , were expressed in Escherichia coli . Recombinant AgaAJB07 showed a 2.9-fold and 22.6-fold increase in k cat with a concomitant increase of 2.3-fold and 16.3-fold in K m in the presence of 0.5 mM ZnSO 4 and 30.0 mM Pb(CH 3 COO) 2 , respectively. Recombinant AgaAHJG4 showed apparent optimal activity at pH 8.0 in McIlvaine or Tris–HCl buffer and 9.5 in glycine–NaOH or HCl–borax–NaOH buffer, retention of 23.6% and 43.2% activity when assayed at 10 and 20 °C, respectively, and a half-life of approximately 2 min at 50 °C. The activation energies for p -nitrophenyl-α- d -galactopyranoside hydrolysis by AgaAJB07 and AgaAHJG4 were 71.9 ± 0.8 and 48.2 ± 2.0 kJ mol −1 , respectively. Both AgaAJB07 and AgaAHJG4 exhibited transglycosylation activity, but they required different acceptors and produced different compounds. Furthermore, potential factors for alkaline and multiple pH optima and low-temperature adaptations of AgaAHJG4 were presumed. [ABSTRACT FROM AUTHOR]

β-D-xylosidases are hemicellulases that catalyze the release of xylose units from short xylooligosaccharides. Their activity is the rate-limiting step in xylan hydrolysis. A major application of these enzymes is as eco-friendly biobleaching agents in the pulp and paper industry, where thermostable, alkaliphilic xylosidases are much preferred. Hence, the isolation and characterization of new thermo-alkaliphilic xylosidases from nature, or re-engineering existing ones is of importance for industrial applications. The thermophilic bacterium Geobacillus thermoleovorans IT-08 produces a meso-thermophilic β-D-xylosidase (EC 3.2.1.37), which has a unique primary structure compared to other xylosidases with only 32-35% amino acid sequence identity. Here, we describe the role of Asp121 in modulating the enzyme's pH-activity profile. Mutation of this residue into Val121 or Asn121 increased its pH optimum by 3 pH units with a significant decrease in its activities. Furthermore, all mutants showed a significant 40 °C increase in optimum temperature compared to wild-type enzyme. This study thus illustrates the importance of the p K a modulator residue Asp121 in shifting the pH- and temperature-activity profile of the enzyme. Changing Asp/Asn residues in enzymes, especially when the residue is located near the catalytic site, may be a useful strategy for adapting the enzyme to a high pH and high temperature environment. [ABSTRACT FROM AUTHOR]

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase. [ABSTRACT FROM AUTHOR]

Struvite (MgNH4PO4.6H2O) is one of phosphate minerals, commonly forms into aqueous solutions. It can be precipitated as mineral deposits for optimization of phosphate recovery based on the pH optimum, molar ratio and temperature levels. This paper presents results of a study on the struvite precipitation under the influence of pH variation, at optimized molar ratio and temperature, which were calculated from an experimental design methodology. Based on the methodology, a laboratory prepared struvite, made by mixing solutions to NH4OH, MgCl2 and H3PO4 for a molar ratio of 1: 2: 1 in a 500 mL volume of batch stirred crystallizer at room temperature. The crystallization was done at 200 rpm and the pH variation was adjusted to 8, 9 and 10 with KOH for a time of 70 minutes. The resulting crystals were filtered and dried at room temperature for 48 h and subsequently stored for further analysis. Material characterisasion of the crystals was conducted using XRPD Rietveld method of mineralogical composition. SEM equipped by EDX was employed for investigation of morphology and elemental composition of the crystals obtained. During the experiment, struvite crystals were firstly nucleated and subsequently developed at major value. The increase in pH is assumed to convert some of the struvite phase into struvite (K) and minor sylvite (KCl). It demonstrates that Visual MINTEQ can be employed to estimate the mineral formation out the synthetic solutions.

The results of BLA ST analyses of partial nucleotide sequence of 16S ribosomal RNA gene of newly isolated strain N 6.1, possessing D-aminoacylase activity, revealed maximum o f 83 % identity with Bacterial and Archaeal databases, indicating that the isolated bacteria belongs to a new species and even the new genus. The enzyme was more than 850 times purified with 2.7 % overall yield as a result o f 4 stage purification scheme. The purified D-aminoacylase showed optimal activity at 400C and pH 7.5-7.8. The enzyme retained 50 % of its initial activity when incubated 20 min at 400C, and in these conditions it was more stable at pH 8.5-9.5. [ABSTRACT FROM AUTHOR]

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. 'Deamidation' in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance. [ABSTRACT FROM AUTHOR]

Bacterial sialidases are enzymes that are involved in a number of vital processes in microorganisms and in their interaction with the host or the environment. Their wide application for scientific and applied purposes requires the search for highly effective and non-pathogenic producers. Here, we report the first description of sialidase from Oerskovia paurometabola . The extracellular enzyme preparation was partially purified. The presence of sialidase was confirmed in native PAGE treated with the fluorogenic substrate 4MU-Neu5Ac. Maximum enzyme activity was registered at 37 °C and in the pH range of 4.0-5.5. The influence of metal ions and EDTA was examined. It was demonstrated that EDTA, Mn 2+ and Ba 2+ ions inhibit the sialidase activity to different extent, while Cd 2+ , Fe 2+ and Fe 3+ have stimulating effect on it. These features are studied for the first time concerning sialidase of Oerskovia representative. Cell bound sialidase and sialate aldolase were also established., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Abstract: Anaerobic ammonium oxidation (anammox) is an emerging technology for nitrogen removal that provides a more environmentally sustainable and cost effective alternative compared to conventional biological treatment methods. The objective of this study was to investigate the inhibitory impact of anammox substrates, metabolites and common wastewater constituents on the microbial activity of two different anammox enrichment cultures (suspended and granular), both dominated by bacteria from the genus Brocadia. Inhibition was evaluated in batch assays by comparing the N2 production rates in the absence or presence of each compound supplied in a range of concentrations. The optimal pH was 7.5 and 7.3 for the suspended and granular enrichment cultures, respectively. Among the substrates or products, ammonium and nitrate caused low to moderate inhibition, whereas nitrite caused almost complete inhibition at concentrations higher than 15mM. The intermediate, hydrazine, either stimulated or caused low inhibition of anammox activity up to 3mM. Of the common constituents in wastewater, hydrogen sulfide was the most severe inhibitor, with 50% inhibitory concentrations (IC50) as low as 0.03mM undissociated H2S. Dissolved O2 showed moderate inhibition (IC50 =2.3–3.8mgL−1). In contrast, phosphate and salinity (NaCl) posed very low inhibition. The suspended- and granular anammox enrichment cultures had similar patterns of response to the various inhibitory stresses with the exception of phosphate. The findings of this study provide comprehensive insights on the tolerance of the anammox process to a wide variety of potential inhibiting compounds. [Copyright &y& Elsevier]

Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. The pH at which the enzyme is maximally active is determined by the pKs of the active site residues, which are modulated by several factors including the change in electrostatics in its vicinity. As the acidic xylanases are important in food and animal feed industries, electrostatic interactions are introduced in Bacillus circulans xylanase to shift their pH optima towards the acidic side. Arg substitutions are made to modulate the pKas of the active site residues. Neutral residues are substituted by Arg in such a way that the substituted residue can make direct interaction with the catalytic residues. However, the mutations with other titratable residues (Asp, Arg, Lys, His, Tyr, and Ser) present in between the catalytic sites and the substituted sites are avoided. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards the acidic side by 0.5-1.5 unit. Molecular dynamics simulation of the mutant V37R reveals that the decrease in activity is due to the increase in distance between the substrate oxygen atoms and catalytic glutamates. [ABSTRACT FROM AUTHOR]

Abstract: Introduction or disruption of long-range electrostatic interactions can be an effective way to change the pK as of catalytic residues and the pH optima of enzymes. In particular, shifting the pH optima toward the acidic or basic limb is an important issue for industrial applications of xylanases, e.g., for the paper or food industries. Here, we suggest an effective strategy to shift the pH optimum of an enzyme by introducing charged residue. Our strategy is to alter the titration behavior of the strongly interacting catalytic glutamates in Bacillus circulans xylanase by introducing acidic or basic residue in juxtaposition to the natively present acidic residues surrounding the catalytic site, thereby shifting the pH-activity profile. Mutation sites were chosen to be long distances (>8.5Å) away from the catalytic sites. The strategy was verified by site-directed mutagenesis experiments. The results show that the pH optimum can be changed (−0.5 to 1.5unit) by strategically selecting the mutation sites. The strategies developed can effectively be applied to change the pH optima of the families of enzymes harboring acidic residues as catalytic residues. [Copyright &y& Elsevier]

Abstract: For technical, environmental and economical reasons, industrial demands for process-fitted enzymes have evolved drastically in the last decade. Therefore, continuous efforts are made in order to get insights into enzyme structure/function relationships to create improved biocatalysts. Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this review, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members considered as true xylanases compared to the other families because of their high substrate specificity. Based on an exhaustive analysis of the sequences and 3D structures available so far, in relation with biochemical properties, we assess biochemical aspects of GH11 xylanases: structure, catalytic machinery, focus on their “thumb” loop of major importance in catalytic efficiency and substrate selectivity, inhibition, stability to pH and temperature. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses. [Copyright &y& Elsevier]

Abstract: To minimise lipid oxidation and maintain high carotenoid and tocol concentrations in wheat flours and products, fifty-seven accessions, belonging to different Triticum species (Triticum monococcum, Triticum turgidum and Triticum aestivum), were assessed for lipoxygenase activity. The highest enzymatic activity was observed in T. aestivum (8.02±0.492μmol/min/g DM), followed by T. turgidum (3.48±0.701) and by T. monococcum (0.45±0.072). While the lipoxygenase was consistently high amongst T. aestivum and steadily low amongst T. monococcum samples, the T. turgidum accessions clustered in three different groups, with low (0.12–0.91μmol/min/g DM), medium (3.10–4.17) and high (5.57–9.51) activity. Enzymatic activity was maximum in the pH range 5–6. LOX activity was higher in the germ (206μmol/min/g DM), than in the bran (13.4) or in the endosperm (3.1). The results demonstrate that the selection of genotypes with low LOX, a factor limiting oxidative degradation, is feasible. [Copyright &y& Elsevier]

The optimal working conditions for enzymes are very much elegant, and their determination is often through experimental approach, which generally is costly and time-consuming. Therefore, it is important to develop methods to use as simple as possible information to predict the optimal working condition for enzymes. Cellulase is a very important enzyme widely used in industries. In this study, we attempted to use a 20-1 feedforward backpropagation neural network to screen 24 amino acid properties related to the primary structure of cellulases as predictors to predict the pH optimum in cellulases. The results show that some predictors can predict the pH, especially amino acid distribution probability. [ABSTRACT FROM AUTHOR]

ginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100-120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime-thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers. [ABSTRACT FROM AUTHOR]

Abstract: Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106–T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of D50 (catalytic nucleophile), whereas those residues are replaced by V109–A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D–S106V–T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces. [Copyright &y& Elsevier]

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175 , a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology. [Display omitted] • TMEM175 is the proton "leak" channel of lysosomes and endosomes • TMEM175 is a highly proton-selective channel that is gated by luminal protons • An endogenous lipid can also activate TMEM175 to trigger lysosomal proton release • TMEM175 sets the lysosomal pH optimum via a classic negative feedback mechanism TMEM175 is a proton-activated proton channel under lysosomal physiological conditions, and it mediates lysosomal proton efflux to maintain a steady-state pH. [ABSTRACT FROM AUTHOR]