Acidic pH Optimum Research Articles

Purification and partial characterization of a thermostable peroxidase isoenzyme from Bambara groundnut (Vigna subterranea L. Verdc) seedlings

Thermostable isoperoxidases, especially peroxidase A6, have already been found in seedlings of Vigna sp, indicating their potential for biotechnological applications. In this study, peroxidase A6 was purified from the roots of Bambara groundnut (Vigna subterranea L. Verdc) seedlings by a combination of gel filtration on Sephadex G-100, heat treatment, and ion exchange chromatography on CM cellulose and DEAE cellulose. The 41-kDa enzyme shows high catalytic efficiency in the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at acidic pH optimum and the reduction of H2O2. It has an optimum temperature of activity of 60 °C and a calculated activation energy of 221.5 KJ/mol for its thermal inactivation at pH 8. Mg2+ inhibits the enzyme. Ca2+ strongly increases its thermal stability, while Mn2+ and Zn2+ decrease it. The enzyme is inhibited by sodium azide at concentrations above 1 µM, with an IC50 value of about 10 µM. This inhibition, in addition to the RZ value of 2.4 (A403nm/A280nm), confirms the presence of a haem group in the active site, which is common for class III plant peroxidases. Due to its unique catalytic and thermal properties, peroxidase A6 has the potential to be a valuable tool for various biotechnological applications, especially for enzyme immunoassays and clinical diagnosis.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal

Chia gum’s molecular structure with distinctive properties as well as the alginate-based hydrogel’s three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30–50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30–40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.

Stress Factors Affecting the Cryopreservation of Biological Components of Seed Virus for Avian Influenza Vaccine Production

In the past, many studies have been done to cryopreserve biological materials for future vaccine production. Scientists have been using different chemicals as cryoprotectants to preserve their cell lines on which desired viruses can be cultivated. Researchers have always been in search for better molecules to avoid cryoinjury during process of cryopreservation. In the present study, different molecules were evaluated for cryo-potency in preserving master seeds of “Vero cell” line and “Avian Influenza viruses”. Cryoprotectants such as (Dimethyl sulfoxide) DMSO and Glycerol were used in different concentration and evaluated at -80oC and -196oC for different time interval. After 15 days and 30 days of cryopreservation the percentage viability of preserved cells were almost equal at both temperatures whereas, after 60 days, 90 days and 120 days the higher percentage of viability was recorded at -196oC for both Vero cells and Avian Influenza virus.  Different pH levels were set for both samples separately with same time interval and found slightly acidic pH (pH5.5) optimum for cryopreservation of Vero cell line and Neutral pH (pH7) for Avian Influenza virus. DMSO (20%) and Glycerol (40%) showed optimum percentage viability when cryopreserved for 120 days without any ill effect.  

Whole-genome sequence and mass spectrometry study of the snow blight fungus Phacidium infestans (Karsten) DSM 5139 growing at freezing temperatures

Phacidium infestans (synonym Gremmenia infestans) is a significant pathogen that impacts Pinus species across the northern regions of Europe and Asia. This study introduces the genome sequence of P. infestans Karsten DSM 5139 (Phain), obtained through Pacbio technology. The assembly resulted in 44 contigs, with a total genome size of 36,805,277 bp and a Guanine–Cytosine content of 46.4%. Genome-mining revealed numerous putative biosynthetic gene clusters that code for virulence factors and fungal toxins. The presence of the enzyme pisatin demethylase was indicative of the potential of Phain to detoxify its environment from the terpenoid phytoalexins produced by its host as a defense mechanism. Proteomic analysis revealed the potential survival strategies of Phain under the snow, which included the production of antifreeze proteins, trehalose synthesis enzymes, desaturases, proteins related to elongation of very long-chain fatty acids, and stress protein responses. Study of protein GH11 endoxylanase expressed in Escherichia coli showed an acidic optimum pH (pH 5.0) and a low optimum temperature (45 °C), which is reflective of the living conditions of the fungus. Mass spectrometry analysis of the methanol extract of Phain, incubated at − 3 °C and 22 °C, revealed differences in the produced metabolites. Both genomic and mass spectrometry analyses showed the ability of Phain to adapt its metabolic processes and secretome to freezing temperatures through the production of osmoprotectant and cryoprotectant metabolites. This comprehensive exploration of Phain's genome sequence, proteome, and secretome not only advances our understanding of its unique adaptive mechanisms but also expands the possibilities of biotechnological applications.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications. This article is protected by copyright. All rights reserved.

Prolylcarboxypeptidase Alleviates Hypertensive Cardiac Remodeling by Regulating Myocardial Tissue Angiotensin II.

Background Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1-7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro-X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results We established a CRISPR/CRISPR-associated protein 9-mediated global prolylcarboxylpeptidase-knockout and adeno-associated virus serotype 9-mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild-type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II-treated prolylcarboxylpeptidase-knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase-knockout hearts showed upregulated extracellular signal-regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno-associated virus serotype 9-mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase-knockout hearts alleviated angiotensin II-induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno-associated virus serotype 9-mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II-induced cardiac dysfunction. Conclusions Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II-induced hypertrophic remodeling by controlling myocardial angiotensin II levels.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s−1·M−1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.

Engineering the optimum pH of β-galactosidase from Aspergillus oryzae for efficient hydrolysis of lactose

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.

Biochemical characterization of a thermophilic exo-arabinanase from the filamentous fungus Rasamsonia emersonii

Arabinan in plant cell wall constitutes a major source of arabinose and arabino-oligosaccharides in nature. Exo-α-l-1,5-arabinanases release arabinose or arabino-oligosaccharides from arabinan in an exo-acting manner and therefore contribute to arabinan degradation. In this study, an exo-α-l-1,5-arabinanase belonging to GH93 family was identified from the thermophilic filamentous fungus Rasamsonia emersonii. The corresponding encoding gene (Reabn93) was cloned from the R.emersonii genome and heterologously expressed in Pichia pastoris. The purified recombinant ReAbn93 exhibited the maximum activity at 70°C and retained 70% of its activity after incubation at 70°C for 3h ReAbn93 had an acidic pH optimum (pH 4.0) but remained stable over a broad pH range (pH 3-9). The specific activity of ReAbn93 toward linear arabinan under optimal conditions was 466.08Umg-1. Similar to the few other reported GH93 members, ReAbn93 degrades linear arabinan or arabino-oligosaccharides in an exo-acting manner with arabinobiose as the only hydrolytic product. Of note, ReAbn93 possessed remarkably better thermostability and higher specific activity compared to the only reported thermophilic counterpart in GH93, and therefore holds potential in relevant biotechnological applications.

Collar Rot Disease of Betelvine (Piper betle L.) under Coastal Saline Zone of West Bengal

Collar rot is an important disease of betelvine (Piper betle L.) caused by the soil borne fungus Sclerotium rolfsii Sacc. The pathogen was isolated from the infected vines cultivated in the coastal saline agro-climatic zone of West Bengal and characterized for its growth and survival under different pH, temperature and soil moisture. The pathogen was found to prefer acidic pH (optimum at pH 5.0 to 5.5) for its mycelial growth, formation of sclerotia and their germination. The saprophytic colonization ability of the fungus was drastically reduced at pH 7.7 and above. Germination of sclerotia was least at a soil moisture level of 20% of field capacity (FC). The increase in soil moisture increased the rate of sclerotial germination, resulting in quick loss of its viability. The saprophytic colonization ability of S. rolfsii was maximum at 60% of FC, which reduced rapidly above 70% of FC. The optimum temperature for maximum mycelial growth, sclerotial formation and its germination was 30±1°C. Mycelial growth, sclerotial formation, germination and saprophytic colonization ability of the fungus ceased completely at 40±1°C. These specific soil temperature and moisture requirements of the fungus S. rolfsii make the pathogen highly selective for its seasonal incidence and proliferation.

Discovery and characterization of a novel Dyp-type peroxidase from a marine actinobacterium isolated from Trondheim fjord, Norway

A new dye-decolorizing peroxidase (DyP) was discovered through a data mining workflow based on HMMER software and profile Hidden Markov Model (HMM) using a dataset of 1200 genomes originated from a Actinobacteria strain collection isolated from Trondheim fjord. Instead of the conserved GXXDG motif known for Dyp-type peroxidases, the enzyme contains a new conserved motif EXXDG which has been not reported before. The enzyme can oxidize an anthraquinone dye Remazol Brilliant Blue R (Reactive Blue 19) and other phenolic compounds such as ferulic acid, sinapic acid, caffeic acid, 3-methylcatechol, dopamine hydrochloride, and tannic acid. The acidic pH optimum (3 to 4) and the low temperature optimum (25 °C) were confirmed using both biochemical and electrochemical assays. Kinetic and thermodynamic parameters associated with the catalytic redox center were attained by electrochemistry.

Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

Comparison of a fungal and a bacterial laccase for lignosulfonate polymerization

Lignin is the second most abundant biopolymer on earth and accrues in large amounts in the pulp- and paper industry. While currently lignins are mainly used for low-value applications such as energy production, recently the potential of laccases for upgrading lignins has been demonstrated. In this study, two laccases of fungal and bacterial origin were characterized regarding their potential to polymerize lignosulfonate. The laccases MaL1 from Melanocarpus albomyces, and SrLA from Streptomyces rochei were heterologously expressed and showed typical characteristics of laccases, like acidic pH optima for ABTS at pH 4 and 5, respectively, and temperature optima at 50 °C and 80 °C. Polymerization of lignosulfonate with MaL1 led to an almost two-fold increase of the molecular weight, according to size exclusion (SEC) multiangle laser light scattering (MALLS) analysis. In contrast, SrLA showed considerably less activity on lignosulfonates, as measured based on oxygen consumption and SEC-MALLS.

Transformation of low molecular compounds and soil humic acid by two domain laccase of Streptomyces puniceus in the presence of ferulic and caffeic acids.

The two-domain bacterial laccases oxidize substrates at alkaline pH. The role of natural phenolic compounds in the oxidation of substrates by the enzyme is poorly understood. We have studied the role of ferulic and caffeic acids in the transformation of low molecular weight substrates and of soil humic acid (HA) by two-domain laccase of Streptomyces puniceus (SpSL, previously undescribed). A gene encoding a two-domain laccase was cloned from S. puniceus and over-expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography to an electrophoretically homogeneous state. The enzyme showed high thermal stability, alkaline pH optimum for the oxidation of phenolic substrates and an acidic pH optimum for the oxidation of K4[Fe(CN)6] (potassium ferrocyanide) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). Phenolic compounds were oxidized with lower efficiency than K4[Fe(CN)6] and ABTS. The SpSL did not oxidize 3.4-dimethoxybenzoic alcohol and p-hydroxybenzoic acid neither in the absence of phenolic acids nor in their presence. The enzyme polymerized HA—the amount of its high molecular weight fraction (>80 kDa) increased at the expense of low MW fraction (10 kDa). The addition of phenolic acids as potential mediators did not cause the destruction of HA by SpSL. In the absence of the HA, the enzyme polymerized caffeic and ferulic acids to macromolecular fractions (>80 kDa and 10–12 kDa). The interaction of SpSL with HA in the presence of phenolic acids caused an increase in the amount of HA high MW fraction and a two-fold increase in the molecular weight of its low MW fraction (from 10 to 20 kDa), suggesting a cross-coupling reaction. Infrared and solution-state 1H-NMR spectroscopy revealed an increase in the aromaticity of HA after its interaction with phenolic acids. The results of the study expand our knowledge on the transformation of natural substrates by two-domain bacterial laccases and indicate a potentially important role of the enzyme in the formation of soil organic matter (SOM) at alkaline pH values.

Three novel rhamnogalacturonan I- pectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential

Rhamnogalaturonans I (RGI) pectins, which are a major component of the plant primary cell wall, can be recalcitrant to digestion by commercial enzymatic cocktails, in particular during fruit juice clarification process. To overcome these problems and get better insights into RGI degradation, three RGI degrading enzymes (RHG: Endo-rhamnogalacturonase; ABF: α-Arabinofuranosidases; GAN: Endo-β-1,4-galactanase) from Aspergillus aculeatinus were expressed in Pichia pastoris, purified and fully biochemically characterized. All three enzymes showed acidic pH optimum, and temperature optima between 40–50 °C. The Km values were 0.5 mg.ml−1, 1.64 mg.ml−1 and 3.72 mg.ml-1 for RHG, ABF, GAN, respectively. NMR analysis confirmed an endo-acting mode of action for RHG and GAN, and exo-acting mode for ABF. The application potential of these enzymes was assessed by measuring changes in viscosity of RGI-rich camelina mucilage, showing that RHG-GAN enzymes induced a decrease in viscosity by altering the structures of the RGI backbone and sidechains.

Discovery of Highly Active Recombinant PNGase H+ Variants Through the Rational Exploration of Unstudied Acidobacterial Genomes.

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases (PNGases, N-glycanases, EC 3.5.1.52) are indispensable tools in releasing N-glycans from glycoproteins. So far, only a limited number of PNGase candidates are available for the structural analysis of glycoproteins and their glycan moieties. Herein, a panel of 13 novel PNGase H+ candidates (the suffix H+ refers to the acidic pH optimum of these acidobacterial PNGases) was tested in their recombinant form for their deglycosylation performance. One candidate (originating from the bacterial species Dyella japonica) showed superior properties both in solution-phase and immobilized on amino-, epoxy- and nitrilotriacetate resins when compared to currently acidic available PNGases. The high expression yield compared to a previously described PNGase H+, broad substrate specificity, and good storage stability of this novel N-glycanase makes it a valuable tool for the analysis of protein glycosylation.

Metabolic coupling in the co-cultured fungal-yeast suite of Trametes ljubarskyi and Rhodotorula mucilaginosa leads to hypersecretion of laccase isozymes

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3–4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment.

An Exochitinase with N-Acetyl-β-Glucosaminidase-Like Activity from Shrimp Head Conversion by Streptomyces speibonae and Its Application in Hydrolyzing β-Chitin Powder to Produce N-Acetyl-d-Glucosamine.

Marine chitinous byproducts possess significant applications in many fields. In this research, different kinds of fishery chitin-containing byproducts from shrimp (shrimp head powder (SHP) and demineralized shrimp shell powder), crab (demineralized crab shell powder), as well as squid (squid pen powder) were used to provide both carbon and nitrogen (C/N) nutrients for the production of an exochitinase via Streptomyces speibonae TKU048, a chitinolytic bacterium isolated from Taiwanese soils. S. speibonae TKU048 expressed the highest exochitinase productivity (45.668 U/mL) on 1.5% SHP-containing medium at 37 °C for 2 days. Molecular weight determination analysis basing on polyacrylamide gel electrophoresis revealed the mass of TKU048 exochitinase was approximately 21 kDa. The characterized exochitinase expressed some interesting properties, for example acidic pH optima (pH 3 and pH 5–7) and a higher temperature optimum (60 °C). Furthermore, the main hydrolysis mechanism of TKU048 exochitinase was N-acetyl-β-glucosaminidase-like activity; its most suitable substrate was β-chitin powder. The hydrolysis experiment revealed that TKU048 exochitinase was efficient in the cleavage of β-chitin powder, thereby releasing N-acetyl-d-glucosamine (GlcNAc, monomer unit of chitin structure) as the major product with 0.335 mg/mL of GlcNAc concentration and a yield of 73.64% after 96 h of incubation time. Thus, TKU048 exochitinase may have potential in GlcNAc production due to its N-acetyl-β-glucosaminidase-like activity.