Major Hydrolysis Products Research Articles

Mass balance, metabolic disposition, and pharmacokinetics of a single IV dose of [14C]CA102N in HT-29 xenograft athymic nude mice

IntroductionCA102N is a novel anticancer drug developed by covalently linking H-Nim (N-(4-Amino-2-phenoxyphenyl methanesulfonamide) to Hyaluronic Acid to target CD44 receptor-rich tumors. The proposed approach seeks to enhance the efficacy and overcome limitations associated with H-Nim, including poor solubility and short half-life.MethodsThe study aimed to evaluate the pharmacokinetics, biodistribution, metabolism, and tumor permeability of [14C] CA102N in xenograft mice following a single intravenous dose of 200 mg/kg. Liquid scintillation counting analysis was used for the pharmacokinetics and mass balance analysis. Metabolite profiling was assessed by HPLC-MS coupled with a radio flow-through detector. Quantitative Whole-Body Autoradiography was used to determine tissue distribution. Concentrations of CA102N and its metabolites were measured using total radioactivity data from urine, feces, and tissue samples.ResultsAbout 94.9% of the administered dose was recovered at 240 h post-dose. The primary route of radioactivity elimination was through urine, accounting for an average of 77% of the dose with around 13.2% excreted in the feces. Tissue distribution showed rapid accumulation within 0.5 h post-administration, followed by a fast decline in most tissues except for the tumor, where slow elimination was observed. CA102N/metabolites exhibited a two-phase pharmacokinetic profile, characterized by an initial rapid distribution phase and a slower terminal elimination, with a half-life (t1/2) of 22 h. The mean maximum concentration (Cmax) of 1798.586 µg equivalents per ml was reached at 0.5 h (Tmax). Most of the radioactivity in plasma was attributed to CA102N, while small-molecule hydrolysis products dominated the excreta and tissue samples. Metabolite profiling revealed two major hydrolysis products: H-Nim-disaccharide and H-Nim-tetrasaccharide. No unchanged [14C] CA102N was detected in urine or feces, suggesting that CA102N undergoes extensive metabolism before excretion.ConclusionThe current data provided valuable insights into the pharmacokinetics, metabolism, and tissue/tumor distribution of CA102N in mice. These findings demonstrated that metabolic clearance is the primary elimination pathway for CA102N and that the drug exhibits tumor retention, supporting its development as an anticancer therapy. Our results provided a strong pharmacological basis for the advancement of CA102N into the clinic.

Functional characterisation of a new halotolerant seawater active glycoside hydrolase family 6 cellobiohydrolase from a salt marsh

Realising a fully circular bioeconomy requires the valorisation of lignocellulosic biomass. Cellulose is the most attractive component of lignocellulose but depolymerisation is inefficient, expensive and resource intensive requiring substantial volumes of potable water. Seawater is an attractive prospective replacement, however seawater tolerant enzymes are required for the development of seawater-based biorefineries. Here, we report a halophilic cellobiohydrolase SMECel6A, identified and isolated from a salt marsh meta-exo-proteome dataset with high sequence divergence to previously characterised cellobiohydrolases. SMECel6A contains a glycoside hydrolase family 6 (GH6) domain and a carbohydrate binding module family 2 (CBM2) domain. Characterisation of recombinant SMECel6A revealed SMECel6A to be active upon crystalline and amorphous cellulose. Mono- and oligosaccharide product profiles revealed cellobiose as the major hydrolysis product confirming SMECel6A as a cellobiohydrolase. We show SMECel6A to be halophilic with optimal activity achieved in 0.5X seawater displaying 80.6 ± 6.93% activity in 1 × seawater. Structural predictions revealed similarity to a characterised halophilic cellobiohydrolase despite sharing only 57% sequence identity. Sequential thermocycling revealed SMECel6A had the ability to partially reversibly denature exclusively in seawater retaining significant activity. Our study confirms that salt marsh ecosystems harbour enzymes with attractive traits with biotechnological potential for implementation in ionic solution based bioprocessing systems.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Effect of di-butyl phosphate on the distribution behavior of uranyl ions in PUREX solvent

ABSTRACT Di-butyl phosphoric acid (HDBP) is one of the major hydrolytic and radiolytic degradation product of tri-n-butyl phosphate (TBP) employed for the extraction of U(VI) and Pu (IV) from the spent nuclear fuel dissolver solution. The presence of HDBP in the solvent phase limits the recovery of actinides during stripping. In view of this, the extraction and stripping behavior of uranyl nitrate in 1.1 M TBP in n-dodecane (n-DD) was studied in presence of di-butyl phosphate (HDBP). The distribution ratio (D) of uranium (VI) in HDBP/n-DD was negligible and the D value increased gradually with an increase in HDBP content in the organic phase. An alternate method was developed for estimation of HDBP in TBP/n-DD system based on uranium metal retention in organic phase. Based on the results it was found that 1 M HDBP in 1.1 M TBP/n-DD retained about 0.63 M of uranium (VI) in the organic phase during stripping. The results were further validated using irradiated solvent. FT-IR spectroscopic investigation also revealed the retention of U(VI) in 1.1 M TBP/n-DD containing HDBP even after several stripping with 0.01 M HNO3.

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or glucose obtainable from cellulose. P. putida EM42, a genome-reduced variant of strain KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose and glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the co-utilization phenotype remained to be understood and the Δgcd strain had a significant growth defect. In this study, we investigated the basis of the simultaneous uptake of the two sugars and accelerated the growth of P. putida EM42 Δgcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion lifted the inhibition of the exogenous β-glucosidase BglC from Thermobifida fusca exerted by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore rapid growth on glucose. The reduced growth rate of the Δgcd mutant was partially compensated by the implantation of heterologous glucose and cellobiose transporters (Glf from Zymomonas mobilis and LacY from Escherichia coli, respectively). Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The pyruvate overproduction phenotype was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions.

Characterization of different chitosanases of Bacillus strains and their application in chitooligosaccharides production.

Chitosanases are potential candidates for chitooligosaccharides (COS) production-based industries, therefore, the discovery of chitosanases having commercial potential will remain a priority worldwide. This study aims to characterize different chitosanases of Bacillus strains for COS production. Six different indigenous Bacillus strains (B. cereus EGE-B-6.1m, B. cereus EGE-B-2.5m, B. cereus EGE-B-5.5m, B. cereus EGE-B-10.4i, B. thuringiensis EGE-B-3.5m, and B. mojavensis EGE-B-5.2i) were used to purify and characterize chitosanases. All purified chitosanases have a similar molecular weight (37 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, other characteristics such as optimum temperature and pH, kinetic parameters (Km and Vmax ), temperature, and pH stabilities were dissimilar among the strains of different Bacillus species and within the same species. Furthermore, chitosanases of all strains were able to successfully hydrolyze chitosan to COS and oligomers of thedegree of polymerization 2-6 were detected with chitobiose and chitotriose as major hydrolysis products. The relative yields of COS were in a range of 19%-31% and chitosanase of B. thuringiensis EGE-B-3.5m turned out to be the best enzyme in terms of its characteristics and COS production potential with maximum relative yield (31%). Results revealed that Bacillus chitosanases could be used directly for efficient bioconversion of chitosan into COS and will be valuable for large-scale production of biologically active COS.

Evaluation of Electrospun PCL-PLGA for Sustained Delivery of Kartogenin.

In this study, kartogenin was incorporated into an electrospun blend of polycaprolactone and poly(lactic-co-glycolic acid) (1:1) to determine the feasibility of this system for sustained drug delivery. Kartogenin is a small-molecule drug that could enhance the outcome of microfracture, a cartilage restoration procedure, by selectively stimulating chondrogenic differentiation of endogenous bone marrow mesenchymal stem cells. Experimental results showed that kartogenin did not affect the electrospinnability of the polymer blend, and it had negligible effects on fiber morphology and scaffold mechanical properties. The loading efficiency of kartogenin into electrospun membranes was nearly 100%, and no evidence of chemical reaction between kartogenin and the polymers was detected by Fourier transform infrared spectroscopy. Analysis of the released drug using high-performance liquid chromatography–photodiode array detection indicated an abundance of kartogenin and only a small amount of its major hydrolysis product. Kartogenin displayed a typical biphasic release profile, with approximately 30% being released within 24 h followed by a much slower, constant rate of release up to 28 days. Although additional development is needed to tune the release kinetics and address issues common to electrospun scaffolds (e.g., high fiber density), the results of this study demonstrated that a scaffold electrospun from biodegradable synthetic polymers is a suitable kartogenin delivery vehicle.

Degradation Kinetic Study and Mechanistic Interpretation of Hydrolysis of Pidotimod by LC-MS/MS: A QbD Assisted Stability Indicating Method Development

The stability of pidotimod was investigated in this study under ICH-recommended stress degradation conditions. To explore the degradation kinetics of pidotimod, a QbD assisted stability indicating assay method was designed and validated according to ICH Q2 (R1) guidelines. The impacts of hydrogen and hydroxide ions were explored, with a focus on the kinetics of pidotimod hydrolytic degradation to identify the rate laws, which were parameterized using linear regression analysis. The degradation product (DP2) was formed as a major degradation product of hydrolysis. Four known and one unknown degradation product were formed under hydrolysis. Degradation of pidotimod in acid and base degradation follows first-order reaction kinetics while the neutral degradation follows zero-order reaction kinetics. The observed degradation products of pidotimod under hydrolytic degradation were identified by LC-MS/MS analysis and a mechanism of hydrolysis is proposed.

Purification and kinetic properties of a novel β-amylase from Penicillum citrinum AS-9

β-amylase-rich preparation produced by the soil fungal strain Penicillium citrinum AS-9 was completely purified within the succeeding steps; ultrafiltration, acetone fractionation and gel filtration on Sephadex G-150 column. Acetone fractionation recovered the highest β-amylase activity and the 85% acetone fraction exhibited 3.5-fold activity that of the crude enzyme. The column affected 8.6-fold purification for β-amylase, which produced maltose as the major product of starch hydrolysis. The pure enzyme showed a Km value of 17.5 mM and Vmax of 17.5 Umg-1 protein, applying Woolf plot and exhibited its maximum velocity at pH 7.1 and 50°C. In absence of substrate, thermal treatments of the enzyme solution at pH 5.2 and 5.5 had the most adverse effects on the enzyme activity. CaCl2 (1mM) activated the enzyme, while each of Ca2+, Fe3+, cysteine, cystine, I2 and p-chloromercuribenzoate (PCMB) had different inhibitory effects. Maltose as the enzyme product at final concentration of 80 mM strongly inhibited the enzyme.

Saccharification of unripe banana flour using microwave assisted starch degrading enzyme hydrolysis for development of wine and vinegar fermentations

Unripe banana flour (UBF) from Musa (ABB) ‘Kluai Namwa’ was used as the substrate for sugar syrup production by microwave assisted starch degrading enzyme hydrolysis. Results showed that a concentration of 300 g/L of UBF subjected to 800 W microwave power for 2.0 min, with subsequent hydrolysis by a low temperature amylase (iKnowZyme® LTAA) and glucoamylase (iKnowZyme® GA) at 50°C for 9 h yielded highest sugar syrup production at 20 ± 0.89 °Brix of total soluble solids (TSS). The major hydrolysis product from UBF determined by thin-layer chromatography (TLC) was glucose, with reduced amounts of maltose and maltotriose. Fermentation by mixed strains of Saccharomyces cerevisiae produced alcohol content at 13.2 ± 0.07% (w/v) after 10 d at room temperature. Acetic acid fermentation achieved using Acetobacter aceti TISTR 354 by surface culture fermentation (SCF) in a stainless-steel tray chamber yielded 5.10 ± 0.12% (v/v) after cultivation at room temperature for 9 d, corresponding to standard commercial vinegar products at over 4.0%. This is the first report detailing production of sugar syrup, wine, and vinegar from UBF, using microwave assisted starch degrading enzyme hydrolysis at 50°C. Results showed that producing an alternative healthy products from natural material could be feasible with added value through biotechnological processes.

Probing the Fate of Different Structures of Beta-Lactam Antibiotics: Hydrolysis, Mineral Capture, and Influence of Organic Matter

Beta-lactam antibiotics, which are used extensively in human and veterinary applications, are commonly detected in surface waters. To examine how the distinct structures of different generations of beta-lactam antibiotics can influence their persistence or degradation in environmental aqueous media, we examined the fate of two penams (amoxicillin and cloxacillin) and two cephems (cephalexin and ceftriaxone) at pH 5.0 and pH 7.0. By contrast to the lack of hydrolysis of the penam antibiotics at both pHs, we observed hydrolysis of cephalexin at pH 7.0 (t1/2 = 12 d) and ceftriaxone at pH 5.0 (t1/2 = 2.8 d). Using high-performance liquid chromatography coupled with a diode array detector or a high-resolution mass spectrometer, we were able to confirm thiotriazinone and 3-desacetyl cefotaxime as major hydrolysis products of ceftriaxone, and propose the hydrolytic cleavage of the benzene and cephem moieties from cephalexin. In addition, we studied the effects of smectite clay particles suspended in solutions without or with dissolved organic matter. The adsorption capacity of the clay was 4- to 9-fold higher at pH 7.0 than at pH 5.0. Subsequent X-ray diffraction analysis revealed that the antibiotic adsorption was not within the clay interlayer nanopores but occurred primarily on the external clay surfaces. The addition of dissolved organic matter interfered with the adsorption of a cephem antibiotic (ceftriaxone) on the clay, but the adsorption of a penam antibiotic (amoxicillin) remained unaffected. We employed molecular modeling simulations to probe the mechanisms of adsorption on the mineral surface. Our findings offer new insights on how the compound structures can dictate different fates of the beta-lactam class of antibiotics in environmental media.

Identification, heterologous expression and biochemical characterization of a novel cellulase-free xylanase B from the thermophilic bacterium Cohnella sp.A01

A novel xylanase gene of 870bp was isolated and cloned from the thermophilic bacteria Cohnella sp.A01 that encodes an enzyme comprising 290 amino acid residues and belonging to the GH10 family of xylanases. The enzyme was efficiently expressed in Escherichia coli BL21, then purified using one-step chromatography. The recombinant XynB-A01 not only exhibited significant thermostability but also showed to be stable in a broad range of pH (3.0–10.0), the presence of some detergents, and organic solvents. Although XynB-A01 demonstrated significant activity toward both beechwood and oat spelt xylans, it showed a stronger affinity and higher activity toward the former. HPLC analysis of beechwood xylan hydrolysis revealed that xylotetraose, xylobiose, and xylose are the major hydrolysis products. The investigation of XynB-A01 in pulp biobleaching showed effectiveness in the release of reducing sugars and chromophores. Simple purification, stability over a broad range of pH and temperature, cellulase-free character, and the capacity to produce xylooligosaccharides are some of the advantages of the xylanase of Cohnella sp.A01, suggesting that it may be a promising candidate for biobleaching of paper pulps, producing xylooligosaccharides, and applying in other industries.

Depletion kinetics and concentration- and time-dependent toxicity of a tertiary mixture of amitraz and its major hydrolysis products in honeybees

Although amitraz is one of the acaricides most commonly applied within beehives, to date, its time-dependent oral toxicity in honeybees has not been investigated, due to amitraz’s instability in aqueous media. In aqueous media such as honey, amitraz rapidly forms a continuously changing tertiary mixture with two of its major hydrolysis products, DMF and DMPF. The contribution of each hydrolysis product to the overall oral toxicity of this acaricide is not known. Therefore, we aimed to characterize the depletion and formation kinetics of amitraz and its hydrolysis products in 50% sucrose solution provided to caged honeybees, including the calculation of the 50% lethal oral concentration (LC50) of amitraz. We sought to determine the contribution of each component of the mixture to the overall observed toxicity. We also investigated the time- and concentration-dependent toxicity of the amitraz mixture and its hydrolysis products. A novel approach based on the analysis of the areas under the depletion and formation curves of amitraz and its hydrolysis products revealed that DMPF, amitraz and DMF accounted for 92%, 7% and 1% (respectively) of the overall toxicity of the mixture. The chronic oral LC50 of amitraz was 3300 μmol/L, of similar magnitude as that of the non-toxic hydrolysis product DMF. The toxicity of DMPF and the mixture decreased over time; whereas the toxicity of DMF increased over time. Amitraz’s instability in aqueous media and the highly toxic profile of DMPF, suggest that DMPF is the actual toxic entity responsible for amitraz’s toxicity toward honeybees.

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunus segnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg−1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL−1 and 2000 ± 23 μmol min−1 mg−1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

Fluorimetric high-throughput screening method for polyester hydrolase activity using polyethylene terephthalate nanoparticles.

Biocatalysis has recently emerged as a powerful and eco-friendly technology in waste plastic recycling, especially for the widely used polyethylene terephthalate (PET). So far, however, a high-throughput screening assay specifically toward PET-hydrolyzing activity has rarely been applied. This hinders the identification of new polyester hydrolases and their variants with adequate activities fulfilling the requirements for industrial applications. This chapter describes the detailed procedure for assaying terephthalate as a major product of enzymatic PET hydrolysis in a 96-well microtiter plate format. Using PET nanoparticles derived readily from waste food packaging as a substrate, an active thermophilic PET hydrolase was clearly distinguished from an inactive variant by a Fenton chemistry-mediated fluorimetric detection. The assay uses enzymes in crude cell lysates, obtained by a simple freeze-thaw protocol. The experimental work validates the applicability of this method for screening mutant libraries of novel PET hydrolases and will thus facilitate the identification of promising variants useful for effective plastic waste recycling.

Simple colorimetric screening of the nerve agent VX using gold nanoparticles and a hand-powered extraction device

A screening method for the highly toxic nerve agent VX is required for forensic and clinical analysis. In this study, a simple colorimetric method using gold nanoparticles (AuNPs) was established as an efficient strategy for VX detection by the naked eye. The AuNPs changed from bright red to deep blue on addition of VX under weakly acidic conditions. In the presence of 2-(diisopropylamino)ethanethiol (DAET), which is a major hydrolysis product of VX, AuNP aggregation was observed. This aggregation occurred under weakly alkaline conditions; therefore, the associated color change occurred at a different pH to that induced by VX addition. The presence of DAET could therefore be identified by a simple pH adjustment. In addition, we developed a hand-powered extraction device for on-site primary screening. The developed method and device were applied for the detection of trace levels of VX and DAET in samples taken from simulated crime scenes.

Degradation Pathway Proposal, Structure Elucidation, and In Silico Toxicity Prediction of Dapagliflozin Propane Diol Hydrolytic Degradation Products

Dapagliflozin (DAP) is therapeutic agent for diabetes mellitus type II patients with insufficient glycemic control by exercise and diet. Stability-indicating LC–MS method was developed and validated on C8 column and method was able to separate DAP and three major hydrolytic degradation products. Structure elucidation of three major identified hydrolytic degradation products (DPs) and establishment of degradation pathway were executed with the combine application of liquid chromatography-positive mode electrospray ionization tandem mass spectrometry and high-resolution mass spectrometry. Further ProTox-II was used to predict in silico toxicity for each DPs and compared with DAP.

Endoglucanase Produced by Bacillus subtilis Strain CBS31: Biochemical Characterization, Thermodynamic Study, Enzymatic Hydrolysis, and Bio-industrial Applications

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. Here, homogeneous endoglucanase GluCB31 from Bacillus subtilis subsp. inaquosorum CBS31 was studied. GluCB31 was purified to 17.68-fold with an 8.33% yield and a specific activity of 1066.37 U/mg. Biochemical properties of GluCB31 were performed and the results are as follows; molecular mass of 35 kDa with an optimum pH at 7.5 and temperature at 50°C. GluCB31 was immobilized in calcium alginate gel and it exhibited the highest activity at 10°C higher temperature than soluble enzyme, as the entrapment in alginate gel made GluCB31 more stable. Kinetic studies showed the Vmax of 1293.33 ± 2.51 U/mg and Km of 0.0183 mg/mL. Enzymatic activity was activated by Tween-20 (106.7%), Tween-80 (111.6%), Triton X-100 (142.3%), SDS (135.5%), Mg++ (185.7%), Cu++ (167.6%), Zn++ (153.7%), Mn++ (106.3%), Ba++ (181.9%), Ni++ (107.2%) while inhibited by Fe++ (15.8%), β-mercaptoethanol (46.8%), EDTA (54.5%). Enthalpy, free energy, and entropy of activation were calculated to be 38.526 kJmol-1, 44.187 kJmol-1, and -17.518 Jmol-1K-1 respectively. Also, ΔGE-S and ΔGE-T were found to be -10.75 kJmol-1 and -45.92 kJmol-1 respectively. A low ΔS, ΔGE-S, and ΔGE-T values were signified enzyme-catalyzed reaction occurs at a fast rate and the existence of the enzyme in its stable state. Cellobiose was the major end product of hydrolysis. These attributes of GluCB31 demonstrated the diversity of catalytic activities and serve in various biotechnological processes, thus deserve to be developed as a bio-industrial agent.

A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

Cooking Affects Glucosinolate Concentration in Mustard Leaves and Seeds

Glucosinolates in mustard leaves (Brassica juncea L.) and seeds (Brassica nigra L.) were investigated via chemical analysis of their major components and the activity of their hydrolysis products. High-performance liquid chromatography (HPLC) revealed that raw seeds have the highest amount of glucosinolates among the samples tested. Boiling of mustard leaves for 15 minutes significantly reduced the total glucosinolate content (10.94 mmol/g) to up to ~80% in comparison with the raw sample (54.31 mmol/g) which is attributed to thermal degradation of glucosinolates or leaching out to the boiling medium. Similarly, roasting of mustard seeds resulted in a marked decline in total glucosinolate content to 33.70 µmol/g compared with that of raw seed samples (86.18 µmol/g) due to thermal degradation. Liquid chromatography-mass spectrometry (LC-MS) revealed that sinigrin (allyl glucosinolate) is the major glucosinolate in mustard leaves, while the seeds contain sinigrin and gluconapin. Allyl isothiocyanate was identified as the major hydrolytic product using gas chromatography-mass spectrometry (GC-MS). The amount of isothiocyanates were quantified via cyclocondensation reaction and HPLC analysis. Boiling of leaves and roasting of seeds have no significant effect on the total isothiocyanate in comparison with the raw samples. However, the amount of isothiocyanates increases upon hydrolysis in the presence of exogenous myrosinase. Lastly, mustard leaf extracts were found to possess antioxidant properties due to its ability to scavenge free radicals as evidenced by the DPPH assay, which could be attributed to bioactive isothiocyanates inherent in the plant samples.