Isocitrate dehydrogenase (IDH) in oleaginous microorganisms plays an essential role in fatty acid synthesis. We expressed and purified an IDH isozyme from the oleaginous fungus Mortierella alpina ...(MaIDH4) and identified its enzymatic properties. MaIDH4 is NADP+-dependent and has no detectable activity with NAD+. It exerts its maximum activity when Mn2+ is used as a cofactor, followed by Mg2+. The optimum temperatures was 60 °C and 65 °C, and the optimum pH was 8.5 and 9.5, in the presence of Mn2+ and Mg2+, respectively. The catalytic efficiency of MaIDH4 for NADP+ was 16.33 (Mn2+) and 3.86 (Mg2+) μM−1s−1, while that for isocitrate was 3.06 (Mn2+) and 0.65 (Mg2+) μM−1s−1. Characterization of the enzymatic properties of MaIDH4 provides a basis for further understanding lipid synthesis in M. alpina.
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•Expression of an IDH isozyme from Mortierella alpina (MaIDH4) in Pichia pastoris.•MaIDH4 was purified and characterized.•MaIDH4 was thermophilic and basophilic.•The optimum metal ion of MaIDH4 was Mn2+.•MaIDH4 depended only on NADP+.
The present experiment was conducted during rainy (kharif) seasons of 2021, 2022 and 2023 at vegetable research farm, ICAR-Indian Agricultural Research Institute, New Delhi to study okra Abelmoschus ...esculentus (L.) Moench cv. Pusa Sawani, crossed with 3 wild accessions, viz. A. manihot var. tetraphyllus (IC 90476-1); IC-141055 and IC- 141040 of A. moschatus. A total of 27 morphological and 9 biochemical parameters were characterized for parental species and their derived F1 hybrids. Results showed that the hybrids possessed superior and beneficial traits than the parents and had a greater alliance with their wild parent. The mean performance of the quantitative characters and heterosis of the three interspecific derived F1s differed significantly from parents. Almost all biochemical parameters value, except moisture were found high in wild accessions followed by interspecific derived F1 hybrid and lowest in cultivated variety Pusa Sawani. Per cent disease incidence for yellow vein mosaic virus (YVMV) and enation leaf curl virus (ELCV) was 92 and 32, respectively in Pusa Sawani. However, both the wild accessions of A. moschatus namely (IC-141055) and (IC-141040) and their interspecific hybrids recorded no incidence of both the disease.
•High level extracellular expression of bacterial PET hydrolyzing enzymes in Bacillus subtilis.•Boosted expression titer by employing the chaperone-overexpression mutant strain.•First report on ...biochemical properties of BhrPETase, a highly active and super thermostable PET hydrolyzing enzyme.
The environmental threat posed by disposal of plastic wastes has drawn extensive attention in recent years wherein polyethylene terephthalate (PET) constitutes one of the major plastic materials in the wastes. Recycling of PET wastes into reusable materials effectively overcomes its accumulation in the environment and can be achieved by mechanical, chemical, and biological processes. In comparison to the other methods, enzymatic treatment utilizing PET hydrolyzing enzymes (PETases) is environmental-friendly which avoids the use of hazardous chemicals. In this study, we report on the secretory expression in Bacillus subtilis a PETase (BhrPETase) from the bacterium HR29, a close homologue of the leaf-branch compost cutinase (LCC) with 94 % sequence identity. The expression titer of BhrPETase reached 0.66 g/L in an engineered chaperone-overexpression Bacillus subtilis strain, and the biochemical characterization of BhrPETase for the first time revealed its high hydrolyzing activity towards amorphous PET in comparison to two reported PET hydrolyzing enzymes LCC and IsPETase, which were expressed under the same expression conditions in Bacillus subtilis in our study. Most intriguingly, purified BhrPETase displayed a melting temperature as high as 101 °C. To our knowledge it is the most thermostable bacterial PETase characterized so far. The superior activity and thermostability of BhrPETase rendered it one of the most promising PETases for plastic waste recycling and bioremediation applications in the future.
Three β-glucosidases from
ATCC15703, namely, BaBgl1A, BaBgl3A, and BaBgl3B, were overexpressed in
. The recombinant β-glucosidases were sufficiently purified using Ni
affinity chromatography, and ...BaBgl1A exhibited the best purification efficiency with a purification factor of 2.3-fold and specific activity of 71.2 U/mg. Three recombinant β-glucosidases acted on
-nitrophenyl-β-glucopyranoside (
NPβGlc) at around pH 7.0 and 30-50°C. The results of the substrate specificity assay suggested that BaBgl1A acted exclusively as β-1,2-glucosidase, while BaBgl3A and BaBgl3B acted mostly as β-1,3-glucosidase and β-1,4-glucosidase, respectively. The substrate specificity of the three recombinant enzymes was further studied using the ginsenosides Rb1 and Rd as substrates. The results of thin-layer chromatography and high-performance liquid chromatography analyses showed that BaBgl1A exhibited the highest bioconversion ability on Rb1 and Rd, where it hydrolyzed the outer C-3 glucose moieties of Rb1 and Rd into the rare ginsenosides Gypenoside XVII and F
; BaBgl3A exhibited medium bioconversion ability on Rb1, where it hydrolyzed both the outer C-3 and C-20 glucose moieties of Rb1 into Gyp XVII and Rd; and BaBgl3B was not active on Rb1 and Rd. These β-glucosidases will act as new biocatalytic tools for transforming ginsenosides and preparing active glycosides and aglycone.
As an industrially important biological macromolecule, xylanase hydrolyzes xylan to produce xylooligosaccarides (XOS). XOS, with a degree of polymerization (DP) 2 to 4, are important prebiotics used ...as food ingredients. In this study, xylanase (5536 U/g substrate) was produced by Pichia stipitis using corncob and wheat bran mixture under solid state fermentation. Crude xylanase were purified and biochemically characterized. XOS hydrolyzed by crude and purified xylanases were quantified. Molecular weight of the purified enzyme was around 31.6 kDa on SDS-PAGE. Enzyme kinetics showed Km and Vmax values of 4.52 mg/mL and 9.17 μmol/min/mL, respectively. The optimal conditions were pH 6.0 and 50 °C. Xylanase was stable at pH 5–8 for 60 min by retaining 57% activity and at 50 °C for 80 min by retaining 65% activity. Cooper and potassium had no inhibitory effect on xylanase activity. Xylan hydrolysates produced by purified xylanase contained 92% XOS consisting of 14% xylotetroase (DP 4), 49% xylotriose (DP 3) and 29% xylobiose (DP 2). These findings indicate the potential of applying purified xylanase for industrial XOS production.
•Xylanase (activity: 5536 U/g) was produced with Pichia stipitis.•Purified xylanase has a molecular weight of 31.6 kDa.•Kinetics analysis showed superior catalytic efficiency (Kcat/Km = 301 mL/s/mg).•XOS with DP 2 to 4 accounted for 92% in xylan hydrolysates.
Here the recombinant GH10 xylanase (HoXyn10) was investigated for biochemical characteristics and synergistic effect with α-glucuronidase (AnGus67) and α-L-arabinofuranosidase (AnAxh62A) on extracted ...Maso bamboo hemicellulose (BCH) to produce xylooligosaccharides (XOS). The GH10 endo-β-1, 4-xylanase from Trichoderma orientalis EU7–22 is heterologously expressed in Pichia pastoris GS115. The crude HoXyn10 showed three bands on the SDS-PAGE profile and one of the proteins was N-glycosylated. The optimum pH and temperature of HoXyn10 were pH= 6.0 and 65 °C, respectively. It showed a wide range of pH stability and remained more than 80% of optimal activity at pH4.5–8.0 after holding at 40 °C for 60 min. HoXyn10 was highly stable at 50 °C without substrate and the residue xylanase activities were retained 97% and 83% after incubating for 2 h and 18 h, respectively. HoXyn10 displayed high activities and almost the same catalytic efficiency to oat-spelt xylan (OSX), Beechwood xylan (BWX) and wheat arabinoxylan (WAX). Notably, the HoXyn10 exhibited apparent activity on Avicel, but showed no activity on CMC-Na. The hydrolytic products from oligosaccharides (X3-X6) by HoXyn10 were all xylose (X1) and xylobiose (X2). The results from FT-IR and NMR showed the backbone of extracted BCH was (1→4)-linked β-D-xylopyranose and the main side chains were α-L-arabinofuranose residues and 4-O-methyl-D-glucuronic acid residues. Based on the characterized structure, when BCH was synergistically hydrolyzed with HoXyn10, AnGus67 and AnAxh62A using appropriate strategy, the yield of xylooligosaccharide (XOS) and xylose were 50.3% and 14.7%. The results indicated HoXyn10 might be a promising tool for various hemicellulose hydrolysis and Maso bamboo hemicellulose is a promising source for XOS production.
•The optimum pH and temperature of Hoxyn10 were pH= 6.0 and 65 °C.•Hoxyn10 had wide range of pH stability and was extremely stable at 50 °C.•The hydrolytic products from oligosaccharides by Hoxyn10 were all X1 and X2.•The yield of xylooligosaccharide and xylose from BCH were 50.3% and 14.7%.
•A newly discovered type of galactosyltransferase from apple is described.•The enzyme, MdUGT78T2, has dual activity on health-promoting flavonol and anthocyanidin food components.•MdUGT78T2 forms ...quercetin-3-O-galactoside and cyanidin-3-O-galactoside.•Catalytic efficiency is 500-fold higher on quercetin than cyanidin.•MdUGT78T2 is expressed at both the early and late stages of fruit maturation.
Anthocyanidin and flavonol glycosides have been linked to the health-promoting effects associated with apple consumption. However, very few enzymes involved in flavonoid glycosylation have been characterised to date. Here, we present the identification and phylogenetic analysis of 234 putative glycosyltransferases involved in flavonoid biosynthesis, and detail the biochemical and structural characterisation of MdUGT78T2 as a strict galactosyltransferase involved in the formation of quercetin-3-O-galactoside and cyanidin-3-O-galactoside, the major glycoconjugates of flavonoids in apple. The enzyme is also active on other flavonoids but with a lower catalytic efficiency. Our data, complemented with gene expression analysis suggest that MdUGT78T2 synthesises the glycoconjugates at both the early and late stages of fruit development. This newly discovered type of catalytic activity can potentially be exploited for in vitro modification of flavonoids to increase their stability in food products and to modify apple fruits and other commercial crops through breeding approaches to enhance their health benefits.
•Extracellular phytase from A. niger has attractive biochemical properties as an animal feed additive•A new biocatalyst was high thermostability and resistance to acidic pHvalues was produced and ...purified.•Phytase was as able to hydrolyze a large number of phosphate substrates, with high yields.•A great variety of metal ions showed to have a beneficent effect on the enzyme, significantly increasing its catalysis.
In this study, an extracellular phytase produced by Aspergillus niger 7A-1, was biochemically characterized for possible industrial application. The enzyme was purified from a crude extract obtained by solid-state fermentation (SSF) of triticale waste. The extract was obtained by microfiltration, ultrafiltration (300, 100 and 30 kDa) and DEAE-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 89 kDa by SDS-PAGE. The purified enzyme was most active at pH 5.3 and 56 °C, and retained 50% activity over a wide pH range of 4 to 7. The enzymatic thermostability assay showed that the enzyme retained more than 70% activity at 80 °C for 60 s, 40% activity for 120 s and 9% after 300 s. The phytase showed broad substrate specificity, a Km value of 220 μM and Vmax of 25 μM/min. The purified phytase retained 50% of its activity with phosphorylated compounds such as phenyl phosphate, 1-Naphthyl phosphate, 2-Naphthyl phosphate, p-Nitrophenyl phosphate and Glycerol-2-phosphate. The inhibition of phytase activity by metal ions was observed to be drastically inhibited (50%) by Ca++ and was slightly inhibited (10%) by Ni++, K+, and Na+, at 10 and 20 mM concentrations. A positive effect was obtained with Mg++, Mn++, Cu++, Cd++ and Ba++ at 25 and 35% with stimulatory effect on the phytase activity.
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 (Ea, kd, Ed, t1/2, 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‾, CH3COO‾, ClO4‾, CN‾, F‾, HCO3‾, I‾, N3‾, NO3‾ and SCN‾), some anions (C6H5O73−, CO32−, SeO3‾ and SO42−) does not affect enzyme activity. Effect of various chemicals on enzyme activity was also investigated. Km, Vmax, kcat and kcat/Km 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 CO2 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.
Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico ...screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression analysis and activity screening of selected proteins, and (iii) complete biochemical/biophysical characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression analysis and the robotic screening of enzymatic activity provided 8 soluble proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temperature from 20 to 70 °C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (k cat/K 0.5 = 96.8 mM–1 s–1), the most thermostable enzyme with melting temperature 71 °C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temperatures, and a biocatalyst degrading the warfare chemical sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.
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