Alkyl hydroperoxide reductase from Bacillus aquimaris MKSC 6.2 protects Esherichia coli from oxidative stress.

Alkyl hydroperoxide reductase genes (ahpCF) from the soft coral associated Bacillus aquimaris MKSC6.2 have been isolated. The cloned 546 bp ahpC gene encodes a 181 amino acid residues polypeptide. The AhpC belongs to typical 2-Cys peroxiredoxin (Prx) containing conserved peroxidatic cysteine residue (C46 ) required for hydroperoxide reduction and conserved resolving cysteine (C166 ). The isolated 1530 bp ahpF gene encodes a polypeptide of 509 amino acid residues with two conserved C128 HNC131 and C337 PHC340 catalytic residues required for reduction of oxidized-AhpC during catalytic turnover. A survival study with Escherichia coli showed that overexpression of AhpC and AhpF resulted in a total protection against 0.16 mM t-butyl hydroperoxide.

Enzymatic Performance of Aspergillus oryzae α-Amylase in the Presence of Organic Solvents: Activity, Stability, and Bioinformatic Studies.

Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.

Nano Delivery Chitosan-Protein/Hydrolysate of Green Peas Bromelain (PHGPB) Synthesized by Colloidal-Spray Drying Method.

Patients with chronic kidney disease (CKD) suffer persistent decreased kidney function. Previous study of protein hydrolysate of green pea (Pisum sativum) bromelain (PHGPB) has shown promising results as an antifibrotic in glucose-induced renal mesangial culture cells, by decreasing their TGF-ß levels. To be effective, protein derived from PHGPB must provide adequate protein intake and reach the target organs. This paper presents a drug delivery system for the formulation of PHGPB using chitosan as polymeric nanoparticles. A PHGPB nano delivery system was synthesized by precipitation with fixed chitosan 0.1 wt.%, followed by a spray drying process at different aerosol flow rates of 1, 3, and 5 L/min. FTIR results showed that the PHGPB was entrapped in the chitosan polymer particles. Homogeneous size and spherical morphology of NDs were obtained for the chitosan-PHGPB with a flow rate of 1 L/min. Our in vivo study showed that the highest entrapment efficiency, solubility, and sustained release were achieved by the delivery system method at 1 L/min. It was concluded that the chitosan-PHGPB delivery system developed in this study improves pharmacokinetics compared to pure PHGPB.

Sub-chronic toxicity study of green peas protein hydrolysate in rats.

Despite green peas protein hydrolysate benefits, very few studies have described the potential toxicity. The acute oral toxicity of green peas protein which hydrolyzed by bromelain at a single dose of 5000 mg/kg BW has been evaluated in Swiss Webster mice and did not produce treatment-related signs of toxicity or mortality in any of the animals tested during the 14-day observation period. The present study aimed was to evaluate the sub-chronic toxicity effects of Protein Hydrolysate Green Peas Bromelain (PHGPB) in Sprague Dawley rats by the regulations of the Indonesian Food and Drug Supervisory Agency 2013. In the repeated dose 28-day oral toxicity study, the administration of 100 mg/kg, 200 mg/kg, and 400 mg/kg/day of green peas protein hydrolysate per body weight revealed no significant difference (P > 0.05) in body weight change, hematological parameters, relative organ weights, and gross findings compared to the control group. Clinical biochemistry analysis and histopathology examinations of liver and kidney showed slight morphological alteration. The oral sub-chronic toxicity test of PHGPB for 28 days did not induce noticeable signs of toxicity. The no-observed adverse effect level (NOAEL) of PHGPB in the sub-chronic toxicity study was dose of the 200 mg/kg BW. The results of our studies PHGPB indicate a lack of toxicity and support the use of functional foods.

Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58.

We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

Nutrition profile and potency of RGD motif in protein hydrolysate of green peas as an antifibrosis in chronic kidney disease.

OBJECTIVES: Fibrosis is the major cause of chronic kidney injury and the primary etiology in diabetic glomerulosclerosis. The initial study of protein hydrolysate of green peas hydrolyzed by bromelain (PHGPB) considered it to improve kidney function parameters and showed no fibrosis in histopathology features in gentamicin-induced nephrotoxicity rats. In the current study, we aimed to assess the nutrition profile and potency of RGD in PHGPB as antifibrosis in chronic kidney disease (CKD). MATERIALS AND METHODS: Green peas (Pisum sativum) were hydrolyzed by bromelain from pineapple juice to obtain PHGPB. The amino acid content of PHGPB was measured using the UPLC method, while the primary structure used LC-MS/MS. Bioinformatic analysis was conducted using the Protease Specificity Predictive Server (PROSPER). The potency of RGD in PHGPB was characterized by determining the levels of Fibronectin (FN) and TGF-ß1 in mesangial SV40 MES 13 cell lines of diabetic glomerulosclerosis. RESULTS: The level of lysine was 364.85 mg/l. The LC-MS/MS data showed two proteins with 4-15 kDa molecular weight originated from convicilin (P13915 and P13919) which were predicted by PROSPER proteolytic cleavage, resulted in RGD in the LERGDT sequence peptide. PHGPB increased SV40 MES 13 mesangial cell proliferation that died from high-glucose levels (diabetic glomerulosclerosis model). PHGPB and RGD reduced the levels of FN and TGF-ß1 in mesangial cell lines of diabetic glomerulosclerosis. CONCLUSION: The nutrition profile and RGD motif in PHGPB show great potential as antifibrosis in CKD.

Biochemical characterization of haloalkane dehalogenases DrbA and DmbC, Representatives of a Novel Subfamily.

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.

Molecular dynamics study to improve the substrate adsorption of Saccharomycopsis fibuligera R64 alpha-amylase by designing a new surface binding site.

Background: Carbohydrate binding module (CBM) and surface binding site (SBS) are two important parts of amylase which respond to the raw starch digestion. They are related to the enzyme ability to adsorb and to catalyze the starch hydrolysis. However, starch processing is still expensive due to the high temperature in the gelatinization step. Therefore, direct starch digestion is more favorable. One of the solutions is to use α-amylase with high starch adsorptivity, which is expected to be capable of digesting starch below the gelatinization temperature. In Indonesia, Saccharomycopsis fibuligera R64 α-amylase (Sfamy R64) is one of the enzymes with the highest activity on starch. However, its raw starch adsorptivity was low. The aim of this study was to propose an in-silico model of Sfamy R64 mutant by introducing a new SBS using molecular dynamics (MD) simulation. Methods: The structural behavior of Sfamy R64 and positive control were studied using MD simulation. Furthermore, the mutants of Sfamy R64 were designed to have a stable SBS which mimics the positive control. The substrate affinity in all systems was evaluated using the molecular mechanics generalized Born surface area (MM/GBSA) method. Results: The stability of a new SBS constructed by seven substitutions and a loop insertion was improved throughout MD simulation. The substrate was consistently bound to the SBS over 55 ns of simulation, as compared to 14 ns in wild-type. The structural behavior of SBS in mutant and positive control was similar. The interaction energies of the positive control, wild-type, and mutant were -17.6, -5.2, and -8.2 kcal/mol, respectively. Conclusion: The enhanced substrate binding in the mutant, due to the existence of a new SBS, suggests the potential of improving starch adsorptivity of Sfamy R64. This result should be useful in developing an enzyme with better substrate adsorption based on the rational computer-aided molecular design approach.

Kidney therapeutic potential of peptides derived from the bromelain hydrolysis of green peas protein.

OBJECTIVES: Kidney disease is a global health problem that needs a solution to its therapy. In the previous study, we found that protein hydrolysate of green peas origin of Indonesia hydrolysed by bromelain (PHGPB) showed improve kidney function in cisplatin-induced nephropathy rats. In this study, we investigated the effect of PHGPB to obtain effective dose that exerts a therapeutic effect on chronic kidney disease (CKD) based on reducing urea and creatinine levels and to elucidate its mechanism of action. MATERIALS AND METHODS: Two sets of experiments were conducted: (1) characteristics and proteomic profile of PHGPB, (2) in vivo test of PHGPB in gentamycin-induced Wistar rats, including urea and creatinine measurements, activities of antioxidant and kidney-related peptides (ANP, COX-1, and renin). RESULTS: PHGPB showed three bands under 10 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and contained 10 identified proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in urea and creatinine levels were found between all PHGPB treatments and positive controls (P<0.01). The lowest levels of urea and creatinine that were validated by high super oxide dismutase (SOD) activity and atrial natriuretic peptide (ANP) level were obtained in the 200 mg/day PHGPB treatment. However, the mean renin level was high and cyclooxygenase-1 (COX-1) level did not exceed positive and negative control levels. CONCLUSION: PHGPB at dose 200 mg/kgBW shows a potential CKD therapeutic effect that is dose-dependent. Higher PHGPB dose corresponds to better effect on kidney function by increasing antioxidant activity and ANP levels in gentamycin-induced Wistar rats.

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption.

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 µM, while that of Sfamy01 was 31 ± 3.9 µM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.

Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K(0.5) for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

Chemical modification of Saccharomycopsis fibuligera R64 α-amylase to improve its stability against thermal, chelator, and proteolytic inactivation.

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.