Substrate Specificity Studies Research Articles

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremelyalkaliphilic bacterium Halalkalibacteriumhalodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60°C. It also presented good stability with a half-life of 3h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46mg/mL and 0.54mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54mg/mL after 4h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Synthetic Sialosides Terminated with 8-N-Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses.

Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged a2-3- and a2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.

Synthetic Sialosides Terminated with 8‐N‐Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses

Sialosides containing C8‐modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid‐interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para‐nitrophenol‐tagged a2–3‐ and a2–6‐linked sialyl galactosides containing C8‐acetamido, C8‐azido, or C8‐amino derivatized N‐acetylneuraminic acid (Neu5Ac). High‐throughput substrate specificity studies showed that the C8‐modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8‐azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8‐acetamido‐modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8‐amino‐modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8‐amino or C8‐acetamido‐modified sialic acid can be used with other sialosides for diagnostic profiling of disease‐causing sialidase‐producing pathogens.

Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

The Catalysis Mechanism of E. coli Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies.

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be -215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π-π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

A Prenyltransferase Participates in the Biosynthesis of Anthraquinones in Rubia cordifolia.

Anthraquinones constitute the largest group of natural quinones, which are used as safe natural dyes and have many pharmaceutical applications. In plants, anthraquinones are biosynthesized through two main routes: the polyketide pathway and the shikimate pathway. The latter primarily forms alizarin-type anthraquinones, and the prenylation of 1,4-dihydroxy-2-naphthoic acid is the first pathway-specific step. However, the prenyltransferase responsible for this key step remains uncharacterized. In this study, the cell suspension culture of Madder (Rubia cordifolia), a plant rich in alizarin-type anthraquinones, was discovered to be capable of prenylating 1,4-dihydroxy-2-naphthoic acid to form 2-carboxyl-3-prenyl-1,4-naphthoquinone and 3-prenyl-1,4-naphthoquinone. Then, a candidate gene belonging to the UbiA superfamily, R. cordifolia dimethylallyltransferase 1 (RcDT1), was shown to account for the prenylation activity. Substrate specificity studies revealed that the recombinant RcDT1 recognized naphthoic acids primarily, followed by 4-hydroxyl benzoic acids. The prenylation activity was strongly inhibited by 1,2- and 1,4-dihydroxynaphthalene. RcDT1 RNA interference significantly reduced the anthraquinones content in R. cordifolia callus cultures, demonstrating that RcDT1 is required for alizarin-type anthraquinones biosynthesis. The plastid localization and root-specific expression further confirmed the participation of RcDT1 in anthraquinone biosynthesis. The phylogenetic analyses of RcDT1 and functional validation of its rubiaceous homologs indicated that DHNA-prenylation activity evolved convergently in Rubiaceae via recruitment from the ubiquinone biosynthetic pathway. Our results demonstrate that RcDT1 catalyzes the first pathway-specific step of alizarin-type anthraquinones biosynthesis in R. cordifolia. These findings will have profound implications for understanding the biosynthetic process of the anthraquinone ring derived from the shikimate pathway.

Molecular cloning, overexpression, characterization, and In silico modelling analysis of a novel GDSL autotransporter-dependent outer membrane lipase (OML) of Pseudomonas guariconensis

The outer membrane lipase (oml) gene, encoding a novel autotransporter-dependent lipase from Pseudomonas guariconensis, was cloned and sequenced. The oml gene has an open reading frame of 1866 bp. It encodes the 621 amino acid autotransporter-dependent GDSL lipase (OML), which has the highest sequence similarity (64.08 %) with the EstA of Pseudomonas aeruginosa (PDB:3kvn.1. A). OML was expressed and purified, which showed a purified band of approximately 70 kDa. The purified enzyme showed maximum activity at pH 9 and 40 °C. Substrate specificity studies and kinetic study by Lineweaver-Burk plot of purified OML showed Km of 1.27 mM and Vmax of 333.33 U/mL with p-nitrophenyl palmitate. The purified enzyme showed good stability in the presence of hexane, methanol, and ethanol, while the presence of the metal ion Mg2+ showed maximum lipase activity. Bioinformatics analysis supported the in vitro findings by predicting enzyme substrate specificity towards long-chain fatty acids and fatty acids with shorter chain lengths. The stability of the interaction of the protein-ligand complex (OML-ricinoleic acid) was confirmed using MDS and castor oil bioconversion using purified OML was confirmed using High-Performance Liquid Chromatography (HPLC).

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Expression and Characterization of a β-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Substrate specificity of polyphenol oxidase and its selectivity towards polyphenols: Unlocking the browning mechanism of fresh lotus root (Nelumbo nucifera Gaertn.).

Polyphenol oxidase (PPO) causes the browning of lotus roots (LR), negatively affecting their nutrition and shelf-life. This study aimed to explore the specific selectivity of PPO toward polyphenol substrates, thus unlocking the browning mechanism of fresh LR. Results showed that two highly homologous PPOs were identified in LR and exhibited the highest catalytic activity at 35 ℃ and pH 6.5. Furthermore, the substrate specificity study revealed (-)-epigallocatechin had the lowest Km among the polyphenols identified in LR, while (+)-catechin showed the highest Vmax. The molecular docking further clarified that (-)-epigallocatechin exhibited lower docking energy and formed more hydrogen bonds and Pi-Alkyl interactions with LR PPO than (+)-catechin, while (+)-catechin entered the active cavity of PPO more quickly due to its smaller structure, both of which enhance their affinity to PPO. Thus, (+)-catechin and (-)-epigallocatechin are the most specific substrates responsible for the browning mechanism of fresh LR.

Heterologous Expression and Characterization of a Thermostable α-L-Rhamnosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 and Its Application in the Biotransformation of Rutin.

An α-L-rhamnosidase gene from Thermoclostridium. stercorarium subsp. thermolacticum DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65°C and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for p-nitrophenyl-α-L-rhamnopyranoside (pNPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60°C, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.

Biochemical properties of purified polyphenol oxidase from bitter leaf (Vernoniaamygdalina)

Polyphenol oxidase which is responsible for oxidative conversion of phenolic compounds to polymers, has continued to attract the attention of scientists. Here, we report the extraction, purification and biochemical properties of polyphenol oxidase (PPO) from bitter leaf (Vernonia amygdalina). The enzyme was purified and concentrated using a non-conventional approach, aqueous two-phase partitioning (ATPS) and the biochemical properties of the purified enzyme were investigated. Substrate specificity studies revealed that the enzyme predominantly exhibits diphenolase activity. The order of substrate preference was catechol > L-DOPA > caffeic acid > L-tyrosine > resorcinol>2-naphthol > phenol. The optimum pH and temperature obtained for the enzyme using catechol as substrate were 5.5 and 50 °C respectively. The estimated Michaelis constant (Km) and maximum velocity (Vmax) for the purified vaPPO using catechol as substrate were 183 ± 5.0 mM and 2000 ± 15 units/mg protein respectively. The catalytic efficiency (Vmax/Km) of the purified vaPPO was 10.9 ± 0.03 min/mg. Na+, K+ and Ba2+ remarkably activated the enzyme and the level of activation was proportional to the concentration. The vaPPO presented stability in the presence of up to 50 mM of the different metal ions tested. In contrast, Cu2+ and NH4+ inhibited the enzyme even 10 mM concentrations. The enzyme was stable in chloroform retaining up to 60% relative activity at 50% (v/v) concentration. There was an increase in the activity (143%) of the enzyme at 30% (v/v) chloroform., revealing that vaPPO could catalyze the substrate more efficiently in 30% (v/v) chloroform. Total loss of enzyme activity was observed at 20% (v/v) concentrations of acetone, ethanol and methanol.In conclusion, the properties of the vaPPO such as its catalysis in the presence of organic solvents, metals and high temperature would be of interest in many biotechnological applications.

Xylose isomerase from Piromyces sp. E2 is a promiscuous enzyme with epimerase activity

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and L-arabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce D-ribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although KM values for D-xylose are comparable at 30 and 60 °C, the kcat/KM is three-fold greater at 60 °C. The purified PirE2_XI shows maximal activity at 65 °C in the pH range of 6.5–7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 °C or 12 h at 60 °C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme.

BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via β-1,4-glycosidic bonds

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via β-1,4 glycosidic bonds, such as β-(Man1 → 4Man), β-(Glc1 → 4Glc) and β-(Xyl1 → 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3–6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 μmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 μmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

Abstract 170: Development of diacylglycerol kinase assays to facilitate isoform specific inhibitor discovery

Abstract Diacylglycerol (DAG) and phosphatidic acid (PA) are two key second messengers in signaling and metabolic pathways. Diacylglycerol kinase (DGK) phosphorylate DAG to produce PA, acting as a central switch between the various signal transduction pathways activated by these second messengers. Ten DGK isoforms (α, β, γ, δ, ε, ζ, η, θ, ι, and κ) have been identified and categorized into five classes based on their structural features. DGKα is highly expressed in several cancers including hepatoma, lymphoma and melanoma, and has been shown to promote cancer survival by positively regulating proliferative activity through several signaling pathways, attracting interest to DGKα as a potential therapeutic target. DGKα and ζ are expressed at high levels in T cells, and shown to promote T cell anergy, pointing to inhibition of these DGK isoforms as a means of enhancing T cell anti-tumor activity and restoring T cells function. Several DGK isoforms have also been implicated in disease contexts such as hypertension, autoimmunity, type II diabetes, epilepsy, and bipolar disorder. Current DGK inhibitor discovery efforts are focused on DGKα and ζ, but to our knowledge no compounds have proceeded to clinical trial-to date. To facilitate these efforts, we have developed a DGK screening panel including compound profiling assays for all DGK isoforms. This is an essential tool for identification of potent and isoform selective inhibitors towards therapeutically relevant DGK isoforms. Further, we present kinetic characterization of all isoforms and lipid substrate specificity studies of DGKs. Citation Format: Safnas F. AbdulSalam, Mia M. Eason, Holly A. Fowle, Anna L. Stuart, Kurumi Y. Horiuchi. Development of diacylglycerol kinase assays to facilitate isoform specific inhibitor discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 170.

Heterologous Expression of a Thermostable α-Galactosidase from Parageobacillus thermoglucosidasius Isolated from the Lignocellulolytic Microbial Consortium TMC7.

α-Galactosidase is a debranching enzyme widely used in the food, feed, paper, and pharmaceuticals industries and plays an important role in hemicellulose degradation. Here, T26, an aerobic bacterial strain with thermostable α-galactosidase activity, was isolated from laboratory-preserved lignocellulolytic microbial consortium TMC7, and identified as Parageobacillus thermoglucosidasius. The α-galactosidase, called T26GAL and derived from the T26 culture supernatant, exhibited a maximum enzyme activity of 0.4976 IU/ml when cultured at 60°C and 180 rpm for 2 days. Bioinformatics analysis revealed that the α-galactosidase T26GAL belongs to the GH36 family. Subsequently, the pET-26 vector was used for the heterologous expression of the T26 α-galactosidase gene in Escherichia coli BL21 (DE3). The optimum pH for α-galactosidase T26GAL was determined to be 8.0, while the optimum temperature was 60°C. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90°C. Furthermore, Ca2+ and Mg2+ promoted the activity of T26GAL while Zn2+ and Cu2+ inhibited it. The substrate specificity studies revealed that T26GAL efficiently degraded raffinose, stachyose, and guar gum, but not locust bean gum. This study thus facilitated the discovery of an effective heat-resistant α-galactosidase with potent industrial application. Meanwhile, as part of our research on lignocellulose degradation by a microbial consortium, the present work provides an important basis for encouraging further investigation into this enzyme complex.

Emerging chemical and biochemical tools for studying 3-O-sulfated heparan sulfate.

Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3-O-sulfated heparan sulfate represents only a small percentage of heparan sulfate from biological sources. However, this subpopulation is closely associated with biological functions of heparan sulfate. The 3-O-sulfated heparan sulfate is biosynthesized by heparan sulfate 3-O-sulfotransferase, which exists in seven different isoforms. This review article summarizes the recent progress in the substrate specificity studies of different 3-O-sulfotransferase isoforms involving the use of homogeneous oligosaccharide substrates and crystal structural analysis. The article also reviews a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to analyze the level of 3-O-sulfated heparan sulfate with high sensitivity and quantitative capability. This newly emerged technology will provide new tools to study the structure and function relationship of heparan sulfate.

Characterization of novel L-asparaginases having clinically safe profiles from bacteria inhabiting the hemolymph of the crab, Scylla serrata (Forskål, 1775).

L-asparaginase (ASNase) is the principal chemotherapeutic agent against different blood cancers. The risks associated with current clinical preparations demand screening for novel ASNases. Accordingly, the study was conducted to shortlist ASNases having clinically safer profiles from a novel niche, namely, microbes in the gut and hemolymph of apparently healthy Scylla serrata. A four-step strategic approach incorporating the essential requirements for clinically safer profiles was followed. The initial step through plate assay showed five (9.61%) potential ASNase producers. The relative prevalence of ASNase producers was higher in hemolymph (13.33%) than gut (4.5%). The positive isolates were identified as Priestia aryabhattai, Priestia megaterium, Bacillus altitudinis, Shewanella decolorationis, and Chryseomicrobium amylolyticum. Quantitative profiles revealed high ASNase production (114.29 to 287.36 U/mL) without any optimization, with an added advantage of the extracellular production. The second step for substrate specificity studies revealed the absence of L-glutaminase and urease activities in ASNases from C. amylolyticum and P. megaterium, the most desirable properties for safe clinical applications. This is the first report of glutaminase and urease-free ASNase from these two bacteria. The third step ensured type II nature of selected ASNases, the targeted form in clinical applications. The fourth step confirmed the activity and stability in human physiological conditions. Altogether, the results revealed two potential ASNases with clinically compatible profiles.

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds.

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 β-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)–beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize β-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.