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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]

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]

In Southeast Asia, Conopomorpha cramerella (Snellen) which is commonly known as the cocoa pod borer (CPB) moth has been identified as the most detrimental pest of Theobroma cacao L. Apart from the various side effects on human health and non‐target organisms, heavily relying on synthetic pyrethroid insecticides to control CPB infestations also increases the environmental contamination risks. Thus, developing biorational insecticides that minimally affect the non‐target organism and environment by targeting the insect growth regulation process is needed to manage the pest population. In insects, juvenile hormones (JH) regulate critical biological events, especially metamorphosis, development and reproduction. Since the physiological roles of JH III vary among different organisms, the biochemical properties, especially substrate specificity and analogue inhibition, may also be different. Therefore, studies on the JH III biosynthetic pathway enzymes in both plants and insects are beneficial to discover more effective analogues. Bioinformatic analysis and biochemical characterization of a NADP+‐dependent farnesol dehydrogenase, an intermediate enzyme of the JH III pathway, from C. cramerella (CcFolDH), were described in this study. In addition, the farnesol analogues that may act as a potent analogue inhibitor for CcFolDH ware determined using in vitro enzymatic study. The phylogenetic analysis indicated that CcFolDH shared a close phylogenetic relationship to the honeybee's short‐chain dehydrogenase/reductase. The 27 kDa CcFolDH has an NADP(H) binding domain with a typical Rossmann fold and is likely a homotetrameric protein in the solution. The enzyme had a greater preference for substrate trans, trans‐farnesol and coenzyme NADP+. In terms of analogue inhibitor inhibition, hexahydroxyfarnesyl acetone showed the highest inhibition (the lowest Ki) compared to other farnesol analogues. Thus, hexahydroxyfarnesyl acetone would serve as the most potent active ingredient for future biorational pesticide management for C. cramerella infestation. Based on the bioinformatic analyses and biochemical characterizations conducted in this research, we proposed that rCcFolDH differs slightly from other reported farnesol dehydrogenases in terms of molecular weight, substrate preference, coenzymes utilization and analogue inhibitors selection. [ABSTRACT FROM AUTHOR]

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk‐clotting enzymes from alternative sources. Trypsin‐like protease by Penicillium roqueforti was produced by solid‐state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert‐type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10–12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin‐like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses. [ABSTRACT FROM AUTHOR]

Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+, Zn2+, and Fe2+. The L-tryptophan was particularly catalyzed compared with D-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08 mM and 0.78 Sec-1, respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila [ABSTRACT FROM AUTHOR]

Raspberry ketone is a primary aroma component of the red raspberry. The glycosylation of this compound is a potential approach used to improve its pharmaceutical properties. In this work, raspberry ketone glycosides are produced in bacteria for the first time. Bacillus licheniformis PI15, an organic solvent‐tolerant glycosyltransferase‐producing strain, was isolated from chemically polluted soil. The cloning and heterologous expression of a glycosyltransferase, which was designated PI‐GT1, in Escherichia coli BL21 resulted in the expression of an active and soluble protein that accounted for 15% of the total cell protein content. Purified PI‐GT1 was highly active and stable over a broad pH range (6.0–10.0) and showed excellent pH stability. PI‐GT1 maintained almost 60% of its maximal activity after 3 H of incubation at 20–40 °C and demonstrated optimal activity at 30 °C. Additionally, PI‐GT1 displayed high stability and activity in the presence of hydrophilic solvents with log P ≤ −0.2 and retained more than 80% of its activity after 3 H of treatment. Supplementation with 10% DMSO markedly improved the glycosylation of raspberry ketone, resulting in a value 26 times higher than that in aqueous solution. The organic solvent‐tolerant PI‐GT1 may have potential uses in industrial chemical and pharmaceutical synthesis applications. [ABSTRACT FROM AUTHOR]

A new bacterial strain producing extracellular cholesterol oxidase (ChOx) was isolated and identified as Castellaniella sp. COX. The ChOx was purified by salting‐out and ion‐exchange chromatography up to 10.4‐fold, with a specific activity of 15 U/mg with a molecular mass of 59 kDa. The purified ChOx exhibited pH 8.0 and temperature 40°C for its optimum activity. The enzyme showed stability over a wide pH range and was most stable at pH value 7.0, and at pH 8.0, it retained almost 86% of its initial activity after 3 h of incubation at 37°C. The enzyme possessed a half‐life of 8 h at 37°C, 7 h at 40°C, and 3 h at 50°C. A Lineweaver–Burk plot was calibrated to determine its Km (0.16 mM) and Vmax (18.7 μmol·mg−1·min−1). The ChOx activity was enhanced with Ca2+, Mg2+, and Mn2+ while it was inhibited by Hg2+, Ba2+, Fe2+, Cu2+, and Zn2+ ions. Organic solvents like acetone, n‐butanol, toluene, dimethyl sulfoxide, chloroform, benzene, and methanol were well tolerated by the enzyme while iso‐propanol and ethanol were found to enhance the activity of purified ChOx. ChOx induced cytotoxicity with an IC50 value of 1.78 and 1.88 U/ml against human RD and U87MG established cell lines, respectively, while broadly sparing the normal human cells. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHAs) are one of the most important biopolyesters produced by vast number of microorganisms that exist in the environment. PHAs gained much attention due to its inherent biodegradable and biocompatible ability along with its physicochemical properties, which are similar to those of conventional petroleum‐based plastics. In the present study, PHA producing bacterial strains was isolated from earthworm. Initially, four bacterial strains were selected based on the intensity of fluorescence from 80 non‐clonal isolates. Bacillus thuringiensis (B.t.) E101 was identified as the bacterium producing highest yield of PHA (3.6 ± 0.04 g L−1) through biochemical and molecular techniques such as 16S rRNA gene sequence analysis. Growth parameters such as carbon, nitrogen sources, and incubation time were optimized with respect to higher PHA production. Both qualitative (Fourier transform infrared spectroscopy and nuclear magnetic resonance) and quantitative (GC–MS) characterizations revealed that the polymer produced by B.t.E101 contains the characteristic peaks for poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer. This is the first report for the occurrence, successful isolation, and characterization of PHA producing Bacillus sp. from earthworm. [ABSTRACT FROM AUTHOR]

Abstract: Enzymes can decrease the environmental and economic load of detergent products by reducing the amount of chemicals used in detergents and by allowing washing at ambient temperatures. In this study, Streptomyces cellulosae AU‐10 (GenBank accession number: MG780240) lipase was purified 7.08‐fold with 68% yield using an aqueous 2‐phase system. The Streptomyces sp. AU‐10 lipase showed maximal activity at pH 9.0 and 40 °C. Hundred percent activities were measured in the pH range from 9.0 to 11.0 for 1 h. The enzyme was also highly stable at 30–50 °C. The values of Km and Vmax were calculated as 0.34 mM and 0.83 mM min−1, respectively. The lipase has high hydrolytic activity for olive oil and sunflower oil. The effect of ethylenediamine tetraacetic acid on the enzyme has shown that the lipase is a metalloenzyme. The activity increased in the presence of Fe2+, Cu2+, and various boron compounds. The enzyme has shown a good stability not only with surfactants but also with oxidizing agents. In addition, activities in the presence of Omo, Ariel, Tursil, Pril, and Fairy were measured as 108.8%, 115.6%, 98.35%, 140.4%, and 107.6%, respectively. Considering its remarkable ability, the S. cellulosae AU‐10 lipase can be considered as a potential additive in the detergent industry. [ABSTRACT FROM AUTHOR]

A lipolytic activity was located in the annular seabream pyloric caeca, from which a digestive lipase (AsDL) was purified. Pure AsDL has an apparent molecular mass of 50 kDa. The purified lipase is thermoactive as it displays its maximal activity on short- and long-chain triacylglycerols at a temperature of 50 °C. The enzyme is alkaline resistant as it retains 90% of its maximal activity when incubated during 1 H at pH 10. No colipase was detected in the annular seabream pyloric caeca. Similar results were reported for the sardine and the gray mullet digestive systems. This is in line with the idea that colipase might have evolved in mammal animals simultaneously with the appearance of an exocrine pancreas. AsDL is a serine enzyme, like all known lipases from different origins. Interestingly, the pure lipase was found to be insensitive to Triton X-100, a synthetic detergent, addition even at a concentration as high as 12 mM. The purified enzyme has potential applications in detergent and food industry because of its thermal activity and alkaline nature. [ABSTRACT FROM AUTHOR]

An extracellular β-glucosidase (), induced by cellulose in the mycelial form of human pathogen fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite ( HAp) adsorption chromatography in batch and Sephacryl S200- HR size exclusion chromatography. The molecular mass of the purified enzyme was estimated to be 197 kDa by size exclusion chromatography with a subunit of 96.8 kDa determined by SDS/ PAGE. The β-glucosidase exhibited optimum catalytic activity at pH 5.5/45 °C and was relatively stable for up to 24 h at 45 °C. Isoelectric focusing displayed an enzyme with a pI value of 4.0. Its activity was inhibited by Fe2+ but not by any other ions or chelating agents. Km and Vmax values of the purified enzyme were 0.012 m m and 2.56 nmol·min−1·mg−1, respectively, using 4-methylumbelliferyl β-D-glucopyranoside (4- MUG) as the substrate and 44.14 m m and 22.49 nmol·min−1·mg−1 when p-nitrophenyl β-D-glucopyranoside ( p- NPG) was used. The purified β-glucosidase was active against cellobioside, laminarin, 4- MUG, and p- NPG and slightly active against 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside but did not hydrolyze 4-methylumbelliferyl β-D-xyloside, 4-methylumbelliferyl β-D-galactopyranoside nor 4-methylumbelliferyl α-D-glucopyranoside. In addition, the enzyme showed transglycosylation activity when it was incubated along with different oligosaccharides. Whether the transglycosylation and cellulase activities function in vivo as a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage of S. schenckii remains to be determined. [ABSTRACT FROM AUTHOR]

Lysoplasmalogen (LyPls)-specific phospholipase D (LyPls- PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1-(1-alkenyl)- sn-glycero-3-phosphate (lysoplasmenic acid). Little is known about LyPls- PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls- PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/ Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions ( pH 8.0 and 50 °C), steady-state kinetic analysis for LyPlsCho yielded Km and kcat values of 13.2 μ m and 70.6 s−1, respectively. The ORF of LyPls- PLD gene consisted of 1005 bp coding a 334-amino-acid (aa) protein. The deduced aa sequence of LyPls- PLD showed high similarity to those of glycerophosphodiester phosphodiesterases ( GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds ( PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls- PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls- PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls- PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles. Data Accessibility Thermocrispum sp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP-01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls-PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively. Enzyme EC number [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: The trachea is essential to respiratory function and is a mechanically and biochemically complex composite tissue. Tissue-engineering approaches to treat tracheal diseases require detailed knowledge of the native mechanical and biochemical properties of the trachea. Although the porcine trachea represents an excellent preclinical model, relevant mechanical and biochemical composition are incompletely characterized.Study Design: Experimental. The mechanical and biochemical properties of 12 intact porcine tracheas were determined to characterize their compliance, as well as the aggregate modulus, bidirectional elastic modulus, hydraulic permeability, and biochemical characteristics of individual cartilage rings.Results: Data demonstrate the glycosaminoglycan content of tracheal rings was (mean ± standard deviation) 190 ± 49 μg/mg. Hydroxyproline content was 8.2 ± 3.2 μg/mg, and DNA content was 1.3 ± 0.27 μg/mg, a four-fold difference between circumferential elastic modulus (5.6 ± 2.0 megapascal [MPa]) and longitudinal composite elastic modulus (1.1 ± 0.7 MPa, P < 0.0001) was also observed. Aggregate modulus (stiffness) of porcine tracheal rings was 1.30 ± 0.28 MPa, and inflationary compliance was 0.00472 ± 0.00188 cmH2 O(-1) .Conclusion: This study presents a comprehensive characterization of the relevant biochemical and mechanical properties of porcine tracheal cartilage, which is considered an excellent candidate for xenogenic tracheal graft and a source for tissue-engineered tracheal reconstruction. The range of parameters characterized in this study agrees with those reported for hyaline cartilage of the airway in other species. These characteristics can be used as quantitative benchmarks for tissue-engineering approaches to treat tracheal disease.Level Of Evidence: NA. Laryngoscope, 126:E325-E331, 2016. [ABSTRACT FROM AUTHOR]

BACKGROUND Polygalacturonases ( PGs) are valuable enzymes of the food industry; therefore it is of great importance to discover new and GRAS PG-producing microbial strains. In this study, PG enzyme produced from a high PG activity producer mutant Aspergillus sojae using wheat bran at the flask scale under pre-optimized conditions of solid-state fermentation ( SSF) was biochemically characterized. RESULTS The crude PG enzyme showed optimum activity in the pH range 4.0-5.0 and was stable in the pH range 3.0-7.0. The optimum temperature for the PG was 40 °C and it retained 99% of its activity at 50 °C. The mutant A. sojae PG could preserve more than 50% of its stability between 25 and 50 °C, both for 30 and 60 min, and was found to be stable in the presence of most of the tested compounds and metal ions. The inactivation energy ( Ed) was determined as 125.3 kJ mol−1. The enthalpy (Δ H*), free energy (Δ G*) and entropy (Δ S*) of inactivation were found to be stable with increasing temperature. CONCLUSION The mutant A. sojae PG could be suitable for the clarification (depectinization) of orange and grape juices and wine. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride. [ABSTRACT FROM AUTHOR]

ABSTRACT A full-length cDNA of a sigma-like glutathione S-transferase (GST) was identified from Hyriopsis cumingii (HcGSTS). The deduced amino acid sequence of HcGSTS was found to comprise 203 amino acid residues and to contain the distinct highly conserved glutathione binding site of N-terminal and the relatively diverse substrate binding site of C-terminal. Alignment analysis and phylogenetic relationship suggested that the HcGSTS is a sigma-class GST. The mRNA of HcGSTS was constitutively expressed in all tested tissues, the strongest expression being in the hepatopancreas. The mRNA expression of HcGSTS was significantly up-regulated ( P < 0.05) in all assessed tissues after stimulation of the mussels with peptidoglycan (PGN) and LPS, the only exception being when the gills were challenged with PGN. The expression of HcGSTS mRNA in kidney and foot was also significantly up-regulated ( P < 0.05) by microcystin-LR. Recombinant HcGSTS exhibited high activity towards the substrate 1-chloro-2,4-dinitrobenzene. The optimal pH was 8.0 and temperature 35 °C. [ABSTRACT FROM AUTHOR]

Pyridoxal-5′-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases. [ABSTRACT FROM AUTHOR]

Cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase (cy- FBP/ SBPase) plays a vital role in gluconeogenesis and in the photosynthetic carbon reduction pathway, and is thus a potential enzymatic target for inhibition of harmful cyanobacterial blooms. Here, we describe the crystal structure of cy- FBP/ SBPase in complex with AMP and fructose-1,6-bisphosphate ( FBP). The allosteric inhibitor AMP and the substrate FBP exhibit an unusual binding mode when in complex with cy- FBP/ SBPase. Binding mode analysis suggested that AMP bound to the allosteric sites near the interface across the up/down subunit pairs C1 C4 and C2 C3 in the center of the tetramer, while FBP binds opposite to the interface between the horizontal subunit pairs C1 C2 or C3 C4. We identified a series of residues important for FBP and AMP binding, and suggest formation of a disulfide linkage between Cys75 and Cys99. Further analysis indicates that cy- FBP/ SBPase may be regulated through ligand binding and alteration of the structure of the enzyme complex. The interactions between ligands and cy- FBP/ SBPase are different from those of ligand-bound structures of other FBPase family members, and thus provide new insight into the molecular mechanisms of structure and catalysis of cy- FBP/ SBPase. Our studies provide insight into the evolution of this enzyme family, and may help in the design of inhibitors aimed at preventing toxic cyanobacterial blooms. Database Structural data have ben submitted to the Protein Data Bank under accession numbers and . Structured digital abstract and by (). [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis isolates were characterized in rhizospheric cotton soils by using acetate selection process from eight different locations in South India. The fact that B. thuringiensis indices were higher in proportion in soil samples taken from Tamil Nadu and Karnataka than from Andhra Pradesh, indicates the abundance of B. thuringiensis populations in the cotton rhizosphere. Biochemical typing of the isolates designated eight local isolates (BtNg13, BtCo1, BtHyb7, BtAm2, BtRm5, BtWr3, BtPl 4, BtN 9), which belong to subspecies kurstaki, the most prevalent subspecies. Toxicity assays on American boll worm larval ( F) populations collected from the Andhra Pradesh, Bangalore and Coimbatore regions, with a susceptible insect strain against different isolates of B. thuringiensis kurstaki spore-crystal mix, revealed distinct susceptibility patterns and specificity. The highest susceptibility was observed in the F populations of Coimbatore, followed by Bangalore and Hyderabad populations, in comparison with the susceptible insect strain. Significant differences were observed ( p < 0.0005 and CD = 5.3975) among Btk local isolates, H. armigera biotypes, Btk spore-crystal mix concentration and their interactions, through the Multifactorial ANOVA analysis. The toxicity of local B. thuringiensis isolates was higher than that of HD-1 (reference B.t.k strain). Indigenous Btk isolates have an enormous potential for the management of H. armigera in terms of development of resistance to HD-1. The present study would serve as a baseline data for future resistance monitoring of B. thuringiensis strains in H. armigera in Southern India. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Pyrimidines are important metabolites in all cells. Levels of cellular pyrimidines are controlled by multiple mechanisms, with one of these comprising the reductive degradation pathway. In the model invertebrate Caenorhabditis elegans, two of the three enzymes of reductive pyrimidine degradation have previously been characterized. The enzyme catalysing the final step of pyrimidine breakdown, 3-ureidopropionase (β-alanine synthase), had only been identified based on homology. We therefore cloned and functionally expressed the 3-ureidopropionase of C. elegans as hexahistidine fusion protein. The purified recombinant enzyme readily converted the two pyrimidine degradation products: 3-ureidopropionate and 2-methyl-3-ureidopropionate. The enzyme showed a broad pH optimum between pH 7.0 and 8.0. Activity was highest at approximately 40 °C, although the half-life of activity was only 65 s at that temperature. The enzyme showed clear Michaelis-Menten kinetics, with a Km of 147 ± 26 μm and a Vmax of 1.1 ± 0.1 U·mg protein−1. The quaternary structure of the recombinant enzyme was shown to correspond to a dodecamer by 'blue native' gel electrophoresis and gel filtration. The organ specific and subcellular localization of the enzyme was determined using a translational fusion to green fluorescent protein and high expression was observed in striated muscle cells. With the characterization of the 3-ureidopropionase, the reductive pyrimidine degradation pathway in C. elegans has been functionally characterized. Structured digital abstract • : 3-ureidopropionase (uniprotkb: ) and 3-ureidopropionase (uniprotkb: ) bind ( ) by blue native page ( ) [ABSTRACT FROM AUTHOR]

Background and Objectives A new chromatographic step for the selective binding of pathological prion proteins (PrPSc) to an affinity ligand, developed and optimized for PrPSc capture and attached to synthetic resin particles (PRDT, USA; ProMetic BioSciences Ltd, Isle of Man, UK) was implemented into the manufacturing process of the solvent/detergent (S/D) treated biopharmaceutical quality plasma Octaplas®. Materials and Methods Pilot batches of Octaplas® with the implemented chromatographic step [labelled as OctaplasLG® (ligand gel)] were manufactured by Octapharma PPGmbH, Vienna, Austria. The biochemical quality was compared directly after manufacturing as well as after 18 months storage. All samples were tested on global coagulation parameters, fibrinogen levels, activities of coagulation factors and protease inhibitors, ADAMTS13 levels, as well as markers of activated coagulation and fibrinolysis. In addition, von Willebrand factor multimeric analysis was performed. Results The incorporation of this novel chromatography into the large-scale routine manufacturing process was shown to be technically feasible and the performance of the column was assessed to be excellent. The biochemical studies showed that Octaplas® and OctaplasLG® produced without and with the new column, respectively, demonstrate an identical biochemical quality. OctaplasLG® remained stable over a period of 18 months stored frozen. A parallel reduction of the S/D virus inactivation step from 4–4·5 to 1–1·5 h led to significantly higher activities of plasmin inhibitor. Conclusion The studies confirmed that the affinity ligand chromatography under the developed conditions can be introduced into the Octaplas® manufacturing process, as a mean to reduce potentially present PrPSc, without hampering the proven quality of this product. [ABSTRACT FROM AUTHOR]

The article discusses the Streptococcus Iniae strains isolated from diseased fish in China and its identification and genetic characterization. It determines the fishes' genetic diversity by pulsed field gel electrophoresis. It mentions that mortalities due to streptococcal infections occurred in seawater cage cultured fish including tilapia. It studies the anatomy of the diseased fishes including the brain, spleen and kidney tissues. It conducts bacterial identification using biochemical and phenotypical tests.

Background and Objectives The most common way to thaw frozen coagulation-active plasma products for transfusion is the use of a water bath with good circulation at 30–37 °C. The aim of this study was to perform an extensive biochemical characterization of the pharmaceutically licenced solvent/detergent-treated plasma, Octaplas®, thawed using the SAHARA-III dry tempering system from the company Sarstedt GmbH, Austria. A regular water bath was used in parallel for comparison. Materials and Methods Six batches Octaplas® with different blood groups were thawed in a water bath or using the SAHARA-III dry tempering system in parallel. Thawed plasma was investigated on screening tests for blood coagulation, as well as on the activities of important coagulation factors and protease inhibitors. In addition, markers of activated coagulation and fibrinolysis were tested and von Willebrand factor multimeric analysis was performed. Results There were neither significant differences in the blood coagulation parameters, coagulation factors, protease inhibitors, nor of markers of activated coagulation and fibrinolysis when Octaplas® thawed by the two different methods was tested. The von Willebrand factor analyses showed no influence on the overall profile of the multimeric pattern when using the SAHARA-III dry tempering system. Conclusion Octaplas® can be thawed using the SAHARA-III dry tempering system without any negative influences on the demonstrated quality of this product. The SAHARA-III dry tempering system enables standardized thawing and warming procedure. Furthermore, tempering of Octaplas® in the emergency unit or operating theatre, where no water baths can be utilized, is safe and can be fully endorsed. [ABSTRACT FROM AUTHOR]

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein–protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70–205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import. [ABSTRACT FROM AUTHOR]

Background and Objectives The ultimate goal was to generate an industrial-scale process suitable to produce a high-yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing. Materials and Methods The new process, which includes three dedicated virus clearance steps, is a streamlined combination of the currently applied and well-established manufacturing procedures. The biochemical characterization is done by standard methods focusing on purity, integrity and functionality of the preparation. Efficacy is demonstrated in vivo by mouse protection testing and in vitro by opsonization and protein A affinity chromatography. Pharmacokinetics in rats is evaluated after a single intravenous dose. The anaphylactoid potential is determined in rats and in guinea pigs, while thrombogenicity is assessed in a rabbit model. The influence of the products on vital functions is tested on dogs, while acute toxicity studies are carried out on mice and rats. Results The biochemical characterization data demonstrate the high purity of monomeric IgG in the product. The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV 10% is at least as good as the reference Gammagard SD. This result is supported by the broad spectrum of antibodies in high titres against bacteria and viruses and the high functional integrity of the IgG molecule (≥ 90% functionally intact IgG) in IGIV 10%. The opsonic activity of all IGIV 10% lots is similar to the one of the reference Gammagard SD. In safety and thrombogenicity studies, no adverse effects of IGIV 10% were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV 10% compared favourably to the reference Gammagard SD. Conclusions The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability. [ABSTRACT FROM AUTHOR]

Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter:: GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89 × 10−5 m and 0.5 × 10−5 m for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66–70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system. [ABSTRACT FROM AUTHOR]

The variability within a collection of 100 isolates of Erwinia collected from various potato cultivars and locations in Algeria was studied using physiological, biochemical and molecular tests. The comparison of their biochemical characteristics with those of the type isolates CFBP 1526 (E. carotovora ssp. atroseptica ), CFBP 2046 (E. carotovora ssp. carotovora ) and CFBP 2048 (E. chrysanthemi ) indicated that all the isolates collected in Algeria belonged to the species E . carotovora . They included 40 typical E. carotovora ssp. carotovora and 14 E. carotovora ssp. atroseptica ; the remaining 46 isolates could not be classified as E. carotovora ssp. atroseptica or ssp. carotovora , even though they were true Erwinia. Amplification of total genomic DNA with the primers Y1 and Y2, specific for E. carotovora , yielded an amplified fragment of the expected size in 99 isolates. The primers Y45 and Y46 specifically amplified a 439-bp DNA fragment in all E. carotovora ssp. atroseptica isolates tested, but not in isolates of the other E. carotovora subspecies or in atypical isolates, as expected from the characteristics of these primers. The digestion patterns of the 99 amplified products with the restriction enzymes Alu I, Hae II, Hpa II and Sau3A I yielded 12 RFLP groups, three of which were undescribed. The 14 isolates of E. carotovora ssp. atroseptica shared a single restriction pattern (RFLP group 1), while the typical isolates of E. carotovora ssp. carotovora and the atypical isolates composed the remaining groups (3, 4, 8–10, 12, 14, 22 and 25–27), reflecting the heterogeneity among these isolates. [ABSTRACT FROM AUTHOR]

A group of ubiquitous small proteins (average 13 kDa) has been isolated from several sensory organs of a wide range of insect species. They are believed to be involved in chemical communication and perception (olfaction or taste) and have therefore been called chemo-sensory proteins (CSPs). Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. We have expressed one of the antennal proteins (CSPMbraA6) in large quantities as a soluble recombinant protein in Escherichia coli periplasm. This 112-residue protein is a highly soluble monomer of 13 072 Da with a pI of 5.5. NMR data (1H and 15N) indicate that CSPMbraA6 is well folded and contains seven α helices (59 amino acids) and two short extended structures (12 amino acids) from positions 5 to 10 and from 107 to 112. Thirty-seven amino acids are involved in β turns and coiled segments and four amino acids are not assigned in the NMR spectra (the N-terminus and the residue 52 in the loop 48–53), probably due to their mobility. This is the first report on the expression and structural characterization of a recombinant CSP. [ABSTRACT FROM AUTHOR]

Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu 2+ , Zn 2+ , and Fe 2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the K m and k cat of the OpTDC2 were 1.08 mM and 0.78 Sec -1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)