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β-Glucosidases for industrial applications are mainly obtained from filamentous fungi. Kretzschmaria zonata is a phytopathogen fungus that produces an arsenal of enzymes with biotechnological potential and this work aimed to produce, purify, and characterize a β-glucosidase from the fungus K. zonata for its application in supplementation of a commercial cocktail for sugarcane bagasse saccharification. The elevated specific activity of β-glucosidase was induced by corn cob, reaching 1.085 U/mg of protein. At the end of all purification steps, a purification factor of 6.52 was reached, with an increase of specific activity from 1.22 U/mg, in the crude extract, to 7.97 U/mg. Concerning pH stability, at pH 4, the pH of maximal β-glucosidase activity, the enzyme was completely stable, with 100% activity after 1 h of incubation, while it kept over 50% activity in the pH range from 2.2 to 6. The optimum temperature was 60 °C and the half-life times were estimated as 307.8 and 10 min, for temperatures of 60 and 70 °C, respectively. The β-glucosidase showed a reduction in relative activity in the presence of 10 mM of manganese sulfate, zinc sulfate, manganese chloride, SDS, and glucose, maintaining 55, 56, 62, 70, and 73% of the relative activity, respectively. The commercial cocktail Multifect® CL supplemented with the K. zonata β-glucosidase enabled the release of 13.89 g/L of glucose and 5.34 g/L of xylose, an increase of 19.8 and 35.5% of glucose and xylose release, respectively, after sugarcane bagasse hydrolysis. [ABSTRACT FROM AUTHOR]

Feather waste produced by livestock and poultry breeding industry is a kind of biomass resource with great application prospect. Current study aims to obtain a feather-degradation bacteria that could efficiently biodegrade feather waste and maintain environmental friendliness. By using the casein agar medium, a novel keratinase production strain Stenotrophomonas sp. Yang-5 was isolated and then identified by subsequent 16S rRNA sequence. Further keratinase production medium was optimized by single factor and orthogonal experiments. Also, crude keratinase was characterized. Consequently, the highest enzyme activity of 161.95 ± 3.65 U/mL was obtained after 48 h of incubation with optimized conditions consisting of reaction temperature 32.5 °C, initial pH 7.5, 0 g/L CaCl2, 50 g/L fructose, and 15 g/L feather. Yang-5 keratinase demonstrated a broad temperature range of 25 °C − 95 °C and optimal catalytic efficiency at pH 9.0. The keratinase had a half-life of more than 120 min at 70° C, indicating satisfactory thermal stability. Furthermore, 0.25 mol/L Mg2+, Mn2+, K+, and Ca2+ could enhance the enzyme activity to some extent and assigned as the metallo-class of keratinase, among of which Mn2+ resulted in the highest improvement by 2-fold activity enhancement. In addition, 5% (v/v) reducing agents including DMSO, DTT and 2-mercaptoethanol all reinforced the enzyme activity by 3–5 folds. To sum up, thermostable alkaline metallo-keratinase from Stenotrophomonas sp. Yang-5 exhibited favorable stability and could serve as a promising candidate for feather waste degradation as well as environmental protection and feed additive industry. Graphical Abst ract: [ABSTRACT FROM AUTHOR]

Enzymes secreted by white-rot fungi, such as laccase, offer a promising solution for treating xenobiotic compounds dangerous to the environment and human health. This study aimed to perform a comprehensive analysis of the tolerance of Pleurotus pulmonarius LBM 105 and its laccase activity toward the pesticides 2,4-D and chlorpyrifos both in vitro and in silico. The fungal strain was able to grow in different concentrations of the pesticides, showing evident morphological alterations. Laccase activity and a 53 kDa electromorph were present in all treatments, showing significant stability with peak activity achieved at a pH of 5.6 and within a temperature range of 50–60 °C. Three laccase genes were mapped, annotated, and characterized from the genome. PplacI obtained better structural validation and affinity energy of − 5.05 and − 7.65 kcal mol−1 with 2,4-D and chlorpyrifos, respectively. The Molecular Mechanics/Poisson-Boltzmann Surface Area analysis at 250 ns confirmed the docking results, revealing the existence of stronger hydrophobic interactions between laccase and chlorpyrifos and highlighting the importance of the Phe341 residue in stabilizing both complexes. Understanding the impact of pesticides on laccase's catalytic function is key to formulating and applying future biotechnological strategies with this enzyme. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70 kDa. They are completely dependent on the coenzyme NADP+ and are inactive towards NAD+. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn2+ and at pH 7.8 in the presence of Mg2+. Its optimal catalytic performance is achieved with both ions at 55 °C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn2+ and Mg2+, respectively, and exhibits optimal catalytic performance at 45 °C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45 °C for 20 min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP+ to NAD+ by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease, which can cause 100% mortality, is one of the main threats to farming Litopenaeus vannamei. We identified seven strains of pathogenic bacteria from ponds with outbreaks of acute hepatopancreatic necrosis at a L. vannamei farm in Zhuhai, China. Among the seven bacteria strains, one strain was isolated from aquaculture water, two strains were isolated from hepatopancreas, and the other four strains were isolated from juvenile shrimp. The results of 16S rRNA sequencing and biochemical identification showed that all seven pathogenic bacteria were Vibrio parahaemolyticus and that they carried the pirA and pirB virulence genes. These bacteria showed ß-hemolysis with translucent rings, and six serotypes were identified. The regression infection results showed that the lethal rates of the three V. parahaemolyticus strains of serotypes O1:KUT and OUT:KUT to L. vannamei were significantly higher than those of the other serotypes. The three V. parahaemolyticus strains had the same growth characteristics, and they all entered the growth plateau at 4 h after inoculation. In addition, the pathogenicity of the three V. parahaemolyticus strains were similar. In the present study, V. parahaemolyticus was identified as one of the pathogenic bacteria causing acute hepatopancreatic necrosis of L. vannamei, and the O1:KUT and OUT:KUT serotypes may be the main pathogenic strains in Zhuhai. These results provided a foundation for the isolation, identification, and pathogenicity study of acute hepatopancreatic necrosis of L. vannamei, providing a reference for the prevention and control direction of L. vannamei disease. [ABSTRACT FROM AUTHOR]

The production of date paste from second-grade date fruits is a fast-growing industrial activity which promotes more sustainable food systems. The industrial date paste process is mainly dependent on the thermal treatments of hydration and drying that precede flesh crushing. These thermal treatments are commonly performed industrially using steam hydration instead of water soaking and convective hot air drying, which are known to be energy-intensive operations leading to high greenhouse gas emissions. The objective of this work was to optimize, on the one hand, the operations of hydration and drying of dates at an industrial scale using a response surface Box–Behnken experimental design in order to reduce the energy consumption and, on the other hand, to assess the biochemical and microstructural properties of date paste produced under optimized conditions. Optimization was performed based on the measurements of sensory attributes, instrumental texture firmness, moisture content, water activity (aw), and color parameters (L*, a*, b*), as well as on the energy savings related to the factors of hydration duration and temperature and time of drying. The optimal conditions to ensure the highest quality of the final product and the lowest energy consumption were 9.6 min of hydration at 80 °C and 3 h of drying at 52.28 °C. The biochemical analysis of the date paste produced under the optimized process showed that it is rich in dietary fibers (9.80 ± 2.10%) and total phenols (261 ± 6.2 mg gallic acid equivalent/100 g of extract). Furthermore, the studied sample exhibited a higher antioxidant potential than the raw date material as a result of the heat-inhibitory effect of oxidases. The obtained results suggest that date paste presents a good source of natural bioactive molecules and could potentially be considered as a functional food ingredient. SEM analysis showed that the microstructural properties of date paste produced under optimal conditions may promote its quality preservation during storage. [ABSTRACT FROM AUTHOR]

Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL−1, Vmax of 155 U mg−1, Kcat 179 s−1, and Kcat/Km 81.06 mg mL−1 s−1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process. [ABSTRACT FROM AUTHOR]

SUMMARY: The conquest of land posed severe problems to plants which they had to cope with by adapting biosynthetic capacities. Adaptations to respond to UV irradiation, water loss, pathogen and herbivore defense, and the earth's pull were essential. Chemical compounds alleviating these problems can be synthesized by the phenylpropanoid pathway, the core of which are three enzymes: phenylalanine ammonia‐lyase (PAL), cinnamic acid 4‐hydroxylase, and 4‐coumaric acid coenzyme A‐ligase (4CL). The genomes of model organisms, Chara braunii as aquatic alga and the two bryophytes Physcomitrium patens and Marchantia polymorpha, were searched for sequences encoding PAL and 4CL and selected sequences heterologously expressed in Escherichia coli for biochemical characterization. Several possible isoforms were identified for both enzymes in Marchantia polymorpha and Physcomitrium patens, while only one or two isoforms could be retrieved for Chara braunii. Active forms of both enzymes were found in all three organisms, although the catalytic efficiencies varied in a wide range. l‐Phenylalanine was accepted as best substrate by all PAL‐like enzymes, despite annotations in some cases suggesting different activities. The substrate spectrum of 4CLs was more diverse, but caffeic and/or 4‐coumaric acids generally were the best‐accepted substrates. Our investigations show that PAL and 4CL, important enzymes for the formation of phenolic compounds, are present and active in extant charophytes and bryophytes as model organisms for the conquest of land. Significance Statement: Upon terrestrialization, plants have profited from an effective phenylpropanoid metabolism enabling them to cope with stressors on land. Essential enzymes, phenylalanine ammonia‐lyase, and 4‐coumaric acid coenzyme A‐ligase are present and active in the charophyte Chara braunii (shown for the first time) and the bryophytes Marchantia polymorpha and Physcomitrium patens and have been characterized on molecular and biochemical levels. [ABSTRACT FROM AUTHOR]

Chitosan oligosaccharide (COS) is a high value-added derivative with intriguing uses in the food, pharmaceutical, and cosmetic industries. Herein, a new chitosanase (CscA) from Kitasatospora setae KM-6054 was cloned and heterologously expressed in Escherichia coli BL21(DE3). CscA showed the highest sequence identity (87.7%) with the chitosanase from Amycolatopsis sp. CsO-2 and was recognized as a new glycoside hydrolase (GH) family 46 chitosanase. The recombinant chitosanase CscA was purified by Ni-NTA Sepharose column and showed a relative molecular weight of 31.07 kDa. CscA had the highest activity (241.39 U/mg) at pH 5.0 and 60 °C. CscA had a wide pH tolerance range, maintaining more than 70% activity in the pH range of 3–10. Furthermore, the enzyme decomposed chitosan to generate chitobiose ((GlcN)2), chitotriose ((GlcN)3), chitotetraose ((GlcN)4), and chitopentaose ((GlcN)5) products. CscA was shown to be a potential chitosanase for the efficient manufacture of COSs from chitosan, which has several uses in food and industry. [ABSTRACT FROM AUTHOR]

AbstractD-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose. [ABSTRACT FROM AUTHOR]

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(smM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(smM) of Vmax,Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes. [ABSTRACT FROM AUTHOR]

Abstract In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg−1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg−1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg−1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5ʹ-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme’s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (K m ) and maximum reaction rate (V max ), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL−1 and 1580.3 ± 183.7 μmol mg−1 protein min−1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.

Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d-luc and Pecan/4-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.

In the current study, an extracellular thermostable lipase from Bacillus sp. strain L2 was produced and purified through two-steps purifications, including ammonium sulfate precipitation and Heparin-Sepharose affinity chromatography. Then, the optimum pH, optimum temperature, thermostability, the effect of metal ions and inhibitors, and substrate specificity towards the natural oils were investigated. Extracellular L2 lipase showed a purification fold of 2.74 and specific activity of 3.54 U/mg towards olive oil as substrate. Furthermore, the purified extracellular L2 lipase had the optimum temperature and pH of 80 °C and pH 7, respectively. The half-lives (t1/2) of L2 lipase at 80 and 85 °C were 150 and 13.43 min, respectively. Moreover, the SDS-PAGE analysis illustrated the single band with a molecular mass of 43 kDa. Moreover, metal ions, including 10 mM concentrations of the Ba2+, Mn2+, Zn2+, Fe3+, Cu2+, and Sr2+, demonstrated inhibitory effects on the L2 lipase activity by decreasing the lipase activity by 100, 18.8, 4.16, 18.86, 100, and 6.25 times. However, the 5 mM concentration of Ca2+ metal ions improved the lipase activity by 1.2 fold. Furthermore, the results after 30 min incubation of L2 lipase with pCMB, PMSF, EDTA, and DTT illustrated that L2 lipase retained 4, 5.3, 5.5, and 26.6% of its initial activity, respectively. The substrate specificity results also illlustrated relative lipase activities of 200, 66.66, 44, 40, 11.33, 9.3, and 13.33% towards sesame oil, coconut oil, rice bran oil, corn oil, sun floweroil, soybean oil, and canola oil, respectively, compared to olive oil. [ABSTRACT FROM AUTHOR]

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35–95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35–95 °C with first order rate constant (kIN) of 5.58 × 10− 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme. [ABSTRACT FROM AUTHOR]

The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme was 10.7-fold higher than the expression level of the native one (0.13 mg mL−1). The recombinant enzyme (His6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg−1. The biochemical properties of the His6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experimental tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality. [ABSTRACT FROM AUTHOR]

Chitin, a polymer of β-1,4-linked N-acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of Shewanella khirikhana JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene SkChi65 from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 °C with 1.0 mM isopropyl-β-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30–50 °C and pH 5.5–8.0 with optima at 40 °C and pH 7.0. The kinetic parameters Km, Vmax, and kcat of SkChi65 towards colloidal chitin were 27.2 μM, 299.2 μMs−1, and 10,203 s−1, respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were N,N'-diacetylchitobiose, and GlcNAc with (GlcNAc)2–6 used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion. [ABSTRACT FROM AUTHOR]

The aim of this research was to explore the chemical composition and antioxidant activities of etheric extracts of Abies marocana. A Soxhlet apparatus was used to extract bioactive molecules from the various parts of the plant. Furthermore, the levels of antioxidant compounds were quantified, while the Gas chromatography was utilized to determine the chemical constituents of the extracted molecules. The extracts were evaluated for their antioxidant properties using the DPPH radical scavenging method and the total antioxidant capacity test. The levels of polyphenols varied across different parts of the plant, ranging from 2.474 ± 0.029 mg.g-1 DM in needles to 4.207 ± 0.008 mg.g-1 DM in twigs. Flavonoids were most abundant in needles 0.140 ± 0.001 mg.g-1 DM and least abundant in cones 0.069 ± 0.007 mg.g-1 DM. Tannins had the highest concentration in twigs 2.608 ± 0.114 mg.g-1 DM, followed by cones 1.948 ± 0.037 mg.g-1 DM and needles 1.512 ± 0.09 mg.g-1 DM. A chromatographic analysis revealed that 56 components were in the samples, with terpene compounds being the most abundant in the different organs. In terms of antioxidant activity, the extract derived from twigs exhibited the strongest antioxidant capacity 49.377 ± 0.371 mg EAA.g-1 DM, followed by cones 35.129 ± 0.084 mg EAA.g-1 DM and needles 13.663 ± 0.084 mg EAA.g-1 DM. Alternatively, the IC50 values for the three organs were found to be in the range of 3844 to 5047.67 µg.mL-1. The results highlight the potential phytopharmaceutical value of A. marocana due to the presence of diverse phyto-components.

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL−1·min−1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of −5.28 kcal/mol, −4.65 kcal/mol, −4.21 kcal/mol, and −5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea. [ABSTRACT FROM AUTHOR]

An approximate revolution synthesis of magnesium oxide (MgO) nanoparticles has been prepared. For plant growth and development, MgO is essential. The effect and efficiency, respectively, in seed germination, seedling growth, and plant growth were also studied. These analyses examined maize with different concentrations and parameters. The concentration of 500 ppm was tested with extreme results in areas such as plant height, protein contents both in-vivo and in-vitro, and MgO effects shown both in shoot (12.83 ± 0.5 cm) and root (5.37 ± 0.5 cm). Maximum confirmations were fixed with the help of MgO NPs characterization through TEM, SEM, FTIR, zeta potential, and X-ray. The effect of MgO NPs showed a significant increase in root and shoot length, and simultaneous in-vivo studies also showed significant results in plant physiological parameters. In effect, the vital performance in germination rate, potential, and index MgO NPs was higher than the control. Moreover, the performance and absorption of MgO NPs was confirmed by physiological characterization with the help of a UV–Vis spectrophotometer applied to the leaves and roots. It was demonstrated that the influence of MgO NPs is positive and potentially can be used for seedling growth and also for plants. It may bolster farming methods, and help maintain high food quality, quantity, and production. [ABSTRACT FROM AUTHOR]

The aim of this research was to explore the chemical composition and antioxidant activities of etheric extracts of Abies marocana. A Soxhlet apparatus was used to extract bioactive molecules from the various parts of the plant. Furthermore, the levels of antioxidant compounds were quantified, while the Gas chromatography was utilized to determine the chemical constituents of the extracted molecules. The extracts were evaluated for their antioxidant properties using the DPPH radical scavenging method and the total antioxidant capacity test. The levels of polyphenols varied across different parts of the plant, ranging from 2.474 ± 0.029 mg.g-1 DM in needles to 4.207 ± 0.008 mg.g-1 DM in twigs. Flavonoids were most abundant in needles 0.140 ± 0.001 mg.g-1 DM and least abundant in cones 0.069 ± 0.007 mg.g-1 DM. Tannins had the highest concentration in twigs 2.608 ± 0.114 mg.g-1 DM, followed by cones 1.948 ± 0.037 mg.g-1 DM and needles 1.512 ± 0.09 mg.g-1 DM. A chromatographic analysis revealed that 56 components were in the samples, with terpene compounds being the most abundant in the different organs. In terms of antioxidant activity, the extract derived from twigs exhibited the strongest antioxidant capacity 49.377 ± 0.371 mg EAA.g-1 DM, followed by cones 35.129 ± 0.084 mg EAA.g-1 DM and needles 13.663 ± 0.084 mg EAA.g-1 DM. Alternatively, the IC50 values for the three organs were found to be in the range of 3844 to 5047.67 µg.mL-1. The results highlight the potential phytopharmaceutical value of A. marocana due to the presence of diverse phyto-components. [ABSTRACT FROM AUTHOR]

Aims: Microorganisms play a vital role in the breakdown of natural organic compounds and are valuable objects for worldwide enzyme production. The aim of this study was to identify favorable production conditions for Bacillus amyloliquefaciens D19 protease, followed by the purification and chemical characterization of this novel enzyme to assess its potential applications in various fields. Methodology and results: In this study, favorable conditions of protease production from B. amyloliquefaciens D19 were determined using a medium containing soluble starch (1.5%), earthworm extract (1.0%), yeast extract (0.5%), NaCl (1.0%), at pH 7.0-8.0, 37 ℃ for 36 h with 150 rpm shaking condition. The protease was purified and had a molecular weight of about 23 kDa. The optimum condition for casein hydrolysis was at 40 ℃ and pH 6.5-7.0 in the presence of 1.0 mM Na+ or 5.0 mM Zn2+. The enzymatic activity was maintained at 75-100% at 30-50 ℃ and in pH 6.0-10.0. The values of Vmax and KM were also determined as 1547 U/mg and 6.33 mg/mL, respectively. Conclusion, significance and impact of study: The identified optimal conditions will serve as the foundation for the production of the 23 kDa B. amyloliquefaciens D19 protease, one of the smallest proteases within the Bacillus genus. Moreover, its notable heat resistance, broad pH tolerance, high substrate catalysis and moderate substrate binding affinity make this enzyme a promising candidate for various applications in the food-feed and brewing industries. [ABSTRACT FROM AUTHOR]

Bacillus species are gram-positive, aerobic, peritrically flagellated and endospore-forming bacteria. They can be found everywhere in the environment, especially in soil (its common habitat), water, dust or in the air. Probiotics, which have beneficial health effects, constitute an important group of Bacillus species. This study aimed to isolate Bacillus from various sources, identify it molecularly and determine its probiotic properties. For this purpose, eight Bacillus subtilis, Bacillus coagulans and Bacillus clausii strains among 58 isolates from fish intestine, soil, ripened cheese and commercial probiotic supplements were identified and their probiotic properties were characterized. Firstly, Bacillus strains were molecularly identified by 16S rRNA PCR analysis. The growth of Bacillus isolates at various temperatures, salt concentrations, and pH levels, as well as tests for esculin hydrolysis, starch hydrolysis, nitrate reduction, and gas generation from glucose, were all investigated to assess the isolates' physiological and biochemical characteristics. In terms of probiotic potential of Bacillus isolates; tolerance of bile salt, cell surface hydrophobicity, auto aggregation, antibiotic susceptibility tests were conducted. In all analyses, strains obtained from food supplements showed high levels of hydrophobicity and auto-aggregation properties, and the highest values following these strains were observed in Bacillus subtilis strains (F1 and S2) isolated from fish intestines and soil, respectively. All strains showed strong growth features in bile salt conditions. It has been determined that antibiotic sensitivity varies depending on the strain. Overall, high sensitivity to tetracycline has been observed. In summary, this study revealed the potential probiotic properties of Bacillus isolates obtained from different sources. The study also compared these probiotic properties with probiotic Bacillus strains isolated from food supplements. [ABSTRACT FROM AUTHOR]

Termites cause a serious menace to wooden structures all over the world. They rely mostly on entozoic fauna residing in their hindgut for the digestion of cellulosic and hemicellulosic materials. One of the ways to control termites is through their gut symbionts. The present study was designed to characterize the hindgut bacteria isolated from Odontotermes obesus and Heterotermes indicola. Furthermore, the growth inhibitory effect of eight tropical plant extracts was investigated to find out potential control agents for these bacterial isolates. The characterization of bacteria was carried out based on their morphology, Gram staining, biochemical and amplification of 16SrRNA gene. Amplified products were sequenced to confirm their relationship with bacterial isolates from termites of other regions. The growth inhibitory effect of ethanolic leaf extracts of eight plants was evaluated in an invitro agar well diffusion method. Qualitative and quantitative phytochemical analysis of the most effective plant was carried out to learn about bioactive agents. The results confirmed the presence of five bacteria from each termite species. The Bacillus cereus, Escherichia coli, and Lysinibacillus fusiformis were common to both termites whereas Lysinibacillus xylanilyticus and Lysinibacillus macrolides were found in O. obesus only and H. indicola harbor Bacillus subtilis and Shigella sonnei in addition to common three ones. Among the plant extracts of Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach, the G. robusta > E. camaldulensis > O. basilicum were found to have growth inhibitory effects with increasing concentrations from 100 to 2000 µg/mL. The biodiversity of the bacterial fauna is important for the biological control of termites. Leaf extracts of these medicinal plants can be used to control termite infestation in an environment-friendly manner to save huge economic loss. [ABSTRACT FROM AUTHOR]

Apis cerana Fab. is one of the popular honeybees species among beekeepers in Indonesia. This species is easy to care for and produces valuable honey products. Honey from A. cerana is abundantly available in traditional and modern markets in Indonesia. The purpose of this study was to identify the primary and secondary metabolites in the honey produced by A. cerana using FTIR-ATR Diamond spectroscopy. Twelve samples of honey from three provinces in Java Island were used in this study. In general, all honey samples contained protein, carbohydrate, water, alcohol, cellulose, alkaloid, tannin, and flavonoid. Variation on primary and secondary metabolites in honey samples was strongly affected by the botanical origin, geographical origin, and the local condition around beekeeping areas where the honeycombs were placed.

In this paper, a Serratia marcescens fibrinolytic protease KD was covalently immobilized onto the electrospun prepared glutaraldehyde (GA)-functionalized chitosan/cellulose acetate membrane nanofibres. Enzyme immobilization has been optimized at some conditions such as different GA values, different crosslinking times, different enzyme and pH values, and different times of immobilization. Results exhibited that the optimized immobilization conditions were obtained in 5.0% GA, after 4 h of crosslinking time, after 8 h immobilization time, using 210 mg protein/g support at pH 9.0. Based on these optimal conditions, the best encapsulation yield (EY) and activity recovery (AR) were obtained about 85% and 121.3%, respectively. The immobilized protease showed a 52% enhancement in protease activity than the free protease in pH 10. Furthermore, results displayed that the Vmax values of free and immobilized enzymes towards casein were gained 0.491 and 0.79 µmol/min, respectively. Moreover, the activity of immobilized protease was retained about 75% after incubation at 60 °C for 180 min at pH 9.0, in which the free protease only preserved about 20% of its primary activity. Results exhibited that the protease-NFs kept nearly 73% of its initial activity after three weeks of storage, while the free protease retained about 20% of its initial activity at the same condition. Results showed that the free protease exhibited 31% clot lysis, whereas the immobilized enzyme exhibited 39% clot lysis. The highest hydrolysis value of both proteases was done 17 and 48% after 4 h at 40 °C, respectively. These results indicated that Chit/CA electrospun nanofibres are excellent membranes for protease immobilization with high application in the digestion of protein waste. [ABSTRACT FROM AUTHOR]

Phosphorus is the second major plant nutrient and the availability of this element to plants is a major challenge due to conversion to insoluble form by chemical reactions with metal cations depending upon the soil pH. Total of sixty eight isolates of phosphate solubilizing bacteria from rhizosphere of mustard grown in different region of Haryana was studied. The isolates were mutagenised by giving treatment of Nitrosoguanidine (50 ug/ml). Three PSB strains (15M, 22M and 25M) and six mutants (15M2, 15M6, 22M28, 22M29, 25M11 and 25M30) were evaluated for their establishment in the rhizosphere, their effect on biomass production in mustard (Brassica juncea). Total bacterial count in rhizosphere increased after 30 and 40 days of sowing while decrease in growth was observed at 60 days of sowing. The phosphate solubilizing bacterial count in the rhizosphere varied from 1 to 24, 2 to 20 and 1 to 11 at 30, 45 and 60 days after sowing. Phosphate uptake also increased upto 11–21% which shows thatmutants had significant effect on increase in plant dry biomass and P-uptake under pot house conditions. [ABSTRACT FROM AUTHOR]

The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01–26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < − 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes. [ABSTRACT FROM AUTHOR]

This work deals with the optimization of an extracellular phospholipase C production by Bacillus cereus (PLCBc) using Response Surface Methodology (RMS) and Box-Behnken design. In fact, after optimization, a maximum phospholipase activity (51 U/ml) was obtained after 6 h of cultivation on tryptone (10 g/L), yeast extract (10 g/L), NaCl (8.125 g/L), pH 7.5 with initial OD (0.15). The PLCBc activity, esteemed by the model (51 U) was very approximate to activity gutted experimentally (50 U). The PLCBc can be considered as thermoactive phospholipase since it showed a maximal activity of 50 U/mL at 60 °C using egg yolk or egg phosphatidylcholine (PC) as substrate. In addition, the enzyme was active at pH 7 and is stable after incubation at 55 °C for 30 min. The application of B. cereus phospholipase C in soybean oil degumming was investigated. Our results showed that when using enzymatic degumming, the residual phosphorus decrease more than with water degumming, indeed, it passes from 718 ppm in soybean crude oil to 100 ppm and 52 ppm by degumming using water and enzymatic process, respectively. The diacylgycerol (DAG) yield showed an increase of 1.2% with enzymatic degumming compared to soybean crude oil. This makes our enzyme a potential candidate for food industrial applications such as enzymatic degumming of vegetable oils. [ABSTRACT FROM AUTHOR]

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg−1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL−1 min−1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications. [ABSTRACT FROM AUTHOR]

Xaa-Pro dipeptidase (XPD, EC 3.4.13.9; also known as prolidase) catalyzes the hydrolysis of the iminopeptide bond in the trans-Xaa-Pro dipeptides (Xaa represents any amino acid except proline), which makes it find wide applications in food, medical and environmental protection fields. In the present study, a novel Xaa-Pro dipeptidase from Aspergillus phoenicis ATCC 14332 (ApXPD) was heterologously expressed and biochemically characterized. Reclassification based on phylogenetic analysis and the version 12.5 MEROPS database showed that this enzyme was the only fungal XPD in the unassigned subfamily that shared the highest sequence identity with Xanthomonas campestris prolidase but not with that from the more related fungal species A. niudulans. As compared with other prolidases, ApXPD also contained a long N-terminal tail (residues 1–63) and an additional region (PAPARLREKL) and used a different arginine residue for dipeptide selectivity. After heterologous expression and partial purification, recombinant ApXPD was highly active and stable over the alkaline range from 8.5 to 10.0, with maximum activity at pH 9.0 and more than 80% activity retained after 1 h incubation at pHs of 8.5–10.0 (55 °C). It also had an apparent optimum temperature of 55 °C and remained stable at 20–30 °C. Moreover, this enzyme was a cobalt-dependent prolidase that only cleaved dipeptides Lys-Pro, Gly-Pro, and Ala-Pro rather than other dipeptides, tripeptides, and tetrapeptides. All these distinct features make A. phoenicis ATCC 14332 XPD unique among currently known prolidases, thus defining a novel Xaa-Pro dipeptidase subfamily. [ABSTRACT FROM AUTHOR]

Essential oils (EOs) are concentrated hydrophobic liquids that originate from plants and contain different bioactive chemicals and volatile substances. Several plant essential oils (PEOs) are obtained from a variety of medicinal plants and have been utilized in folk medicine and traditional pharmacopoeia. They have a long history of usage as antibacterial medicines to treat various human, animal, and plant diseases. The extraction of essential oils frequently involves fractional distillation with a variety of organic solvents. EOs can be used successfully in the food and cosmetics industries in addition to their traditional use as antimicrobial agents. This Special Issue covers various significant PEOs and their individual chemical constituents and biological-pharmaceutical functions. Further information focused on the chemical characterizations, modes of action, and biopharmaceutical properties of PEOs. This Special Issue includes seventeen research papers from different geographical zones. [ABSTRACT FROM AUTHOR]

A novel carbonic anhydrase II (CA II) from erythrocytes of camel (Camelus dromedarius) was purified to homogeneity using affinity chromatography and biochemically characterized. Specific activity of 140.88 U/mg was obtained with 745.17-fold purification and 25.37% yield. The enzyme was a monomer with a lower molecular weight (25 kDa) and lower Zn content (0.50 mol of Zn per mol of protein). The enzyme showed higher optimum temperature (70 °C) and pH (pH 9.0), moreover, it was stable at higher temperatures and strongly alkaline pH as judged by thermodynamic parameters (E a , k d , E d , t 1/2 , D-value, Z-value, ΔH , ΔG and ΔS). The enzyme was inhibited by cations (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Ni2+, Mg2+ and Zn2+) as well as by anions (Br‾, CH 3 COO‾, ClO 4 ‾, CN‾, F‾, HCO 3 ‾, I‾, N 3 ‾, NO 3 ‾ and SCN‾), some anions (C 6 H 5 O 7 3−, CO 3 2−, SeO 3 ‾ and SO 4 2−) does not affect enzyme activity. Effect of various chemicals on enzyme activity was also investigated. K m , V max , k cat and k cat / K m values for 4-NPA were found to be 1.74 mM, 0.0093 U/mL, 0,0039 s−1 and 0,0023 s−1 mM−1, respectively. With these interesting biochemical properties, camel CA II represents promising candidate for harsh industrial applications, in particular, for a successful biomimetic CO 2 sequestration process. • Purification of a novel CA II from camel erythrocytes was carried out. • Camel erythrocytes CA II was biochemically characterized. • CA II showed higher optimum temperature (70 °C) and pH (pH 9.0). • CA II was stable at higher temperatures and strongly alkaline pH. • CA II represents promising candidate for harsh industrial applications. [ABSTRACT FROM AUTHOR]

Black gram is one of the most important short duration grain legume, which contributes significantly towards nutritional security and environmental sustainability. The virus specific primers confirms the presence of mungbean yellow mosaic India virus (MYMIV) in representative samples. A total of 27 cultivated and two wild species were found as highly resistant (HR) to MYMIV and validated through molecular markers. The start codon target (SCoT) markers analysis revealed that the SCoT loci, namely, SCoT‐4 (2200 bp), SCot‐9 (1150/ 1200 bp), SCoT‐15 (1150/1100 bp), SCoT‐16 (700 bp), SCoT‐24 (2500 bp), SCoT‐25 (700 bp), SCoT‐33 (900/1000 bp), and SCoT‐34 (600 bp), were found unique, able to distinguish HR and highly susceptible (HS) genotypes. Biochemical characterization and gene expression profiling revealed the higher expression of antioxidants and R‐genes just after pathogen inoculation indicated the activation of defence mechanism in both cultivated and its wild relatives, which modulates the resistant responses in cultivated and wild accessions. These information will be really helpful in accelerating resistance breeding in black gram. [ABSTRACT FROM AUTHOR]

The discovery and characterization of new alginate lyases with high activity and stability have been the research topic in utilization of alginate biomass. Herein, we characterized a new Thalassotalea algicola -derived polyM-preferred PL6 family alginate lyase TAPL6 with two-domains (catalytic domain NTD and non-catalytic domain CTD). TAPL6 is a bifunctional enzyme that could endolytically degrade polyM, and could exolytically degrade polyG. It could degrade alginate in a hybrid action mode. The specific activities of polyM, polyG and sodium alginate of TAPL6 were 7174 U/mg, 4140.2 U/mg and 414.3 U/mg, respectively. The optimal temperature and pH for TAPL6 are 25 °C and 8.0, respectively. Deletion of CTD resulted in a loss of 99.8%− 98.2% of TAPL6 activity and a decrease in pH adaptability, but greatly improved the activity and temperature stability above 30 °C. The CTD truncation is more resistant to metal ions than TAPL6, and the promotion effect of Ca2+ on the activity is stronger. Deletion of CTD results in a change in the action mode of TAPL6 to an endo-mode. In addition, the integrity of the linker between NTD and CTD is very important for the soluble expression of recombinant protein, and the complete linker is beneficial to the catalytic activity of NTD. This study provides a new tool enzyme for the production of alginate oligosaccharides, and explores the role of the non-catalytic domain CTD in the PL6 family alginate lyases, providing a rational design basis for the PL6 family alginate lyases to change the enzymatic properties and action mode. [Display omitted] • Biochemical characterization of a new PL6 alginate lyase and its domain truncations. • Function of CTD structural domain in the whole enzyme. • Action mode of TAPL6 and the effect of CTD on product distribution and action mode. [ABSTRACT FROM AUTHOR]

The bacterial leaf spot (BLS) is a devastating tomato disease affected by the following Xanthomonas species: X. campestris pv. Vesicatoria, X. euvesicatoria, X. vesicatoria, X. perforans, and X. gardner. Dark lesions and yellow halos on the fruits and foliage are symptoms. The BLS pathogens are widespread in subtropical and tropical areas. Pathogens can be detected using a variety of techniques, most notably a mix of classical and molecular methodologies. Conventional techniques rely on microscopic and culture observation, but Polymerase chain reaction (PCR) and multilocus sequence analysis are obtainable. Control of BLS is problematic because of the infections extensive genetic variety, an absence of durable host resistance, and the ineffectiveness of chemical management. Various bicontrol agents, such as bacteriophage, bacteria and fungi have been documented. Including sustained host resistance is an essential component of continuing integrated BLS management. This review involved state of BLS in tomato comprising its prevalence, pathogen profiles, investigative tools, disease control and resistance development in plant. [ABSTRACT FROM AUTHOR]