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Biotechnology For Biofuels and... Nov 2022Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use...
BACKGROUND
Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use as biotechnological feedstock is still limited.
RESULTS
In order to identify efficient enzymes for polysaccharide breakdown, we have carried out functional screening of metagenomic fosmid libraries from biogas fermenter microbial communities grown on sugar beet pulp, an arabinan-rich agricultural residue, or other sources containing microbes that efficiently depolymerize polysaccharides, using CPH (chromogenic polysaccharide hydrogel) or ICB (insoluble chromogenic biomass) labeled polysaccharide substrates. Seventy-one depolymerase-encoding genes were identified from 55 active fosmid clones by using Illumina and Sanger sequencing and dbCAN CAZyme (carbohydrate-active enzyme) annotation. An around 56 kb assembled DNA fragment putatively originating from Xylanivirga thermophila strain or a close relative was analyzed in detail. It contained 48 ORFs (open reading frames), of which 31 were assigned to sugar metabolism. Interestingly, a large number of genes for enzymes putatively involved in degradation and utilization of arabinose-containing carbohydrates were found. Seven putative arabinosyl hydrolases from this DNA fragment belonging to glycoside hydrolase (GH) families GH51 and GH43 were biochemically characterized, revealing two with endo-arabinanase activity and four with exo-α-L-arabinofuranosidase activity but with complementary cleavage properties. These enzymes were found to act synergistically and can completely hydrolyze SBA (sugar beet arabinan) and DA (debranched arabinan).
CONCLUSIONS
We screened 32,776 fosmid clones from several metagenomic libraries with chromogenic lignocellulosic substrates for functional enzymes to advance the understanding about the saccharification of recalcitrant lignocellulose. Seven putative X. thermophila arabinosyl hydrolases were characterized for pectic substrate degradation. The arabinosyl hydrolases displayed maximum activity and significant long-term stability around 50 °C. The enzyme cocktails composed in this study fully degraded the arabinan substrates and thus could serve for arabinose production in food and biofuel industries.
PubMed: 36371193
DOI: 10.1186/s13068-022-02216-9 -
Environmental Microbiology Feb 2023The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently...
The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.
Topics: Pentoses; Xylose; Arabinose; Pseudomonas putida; Oxidative Stress
PubMed: 36465038
DOI: 10.1111/1462-2920.16296 -
Spectrochimica Acta. Part A, Molecular... Nov 2019FTIR and NMR spectra were measured in parallel for specific two-components mixtures of various proteins with different sugar molecules, such as arabinose, glucose, and...
FTIR and NMR spectra were measured in parallel for specific two-components mixtures of various proteins with different sugar molecules, such as arabinose, glucose, and sucrose. In the FTIR spectra of arabinose with some of these proteins, the bands assigned to the vibrational modes of the CH and COH groups disappeared, and new ones, related to an arabinose-protein CN mode, appeared. Similar changes were observed in the FTIR spectra of lyophilized mixtures of arabinose with different amino acids. In additional FTIR spectra, measured for other protein-sugar mixtures, the bands correlated to the ring modes of arabinose, in the range 1150-1000 cm, disappeared, and two new very strong narrow bands became dominant, indicating ring opening or some kind of arabinose decomposition. Contrary to the prevailing opinion that complexes between sugars and proteins are formed mainly by hydrogen bonds, the IR and NMR spectra of the sugar-protein mixtures studied here suggest that significant chemical reactions also take place between the interacting sugar and the protein. Two types of sugar-protein chemical reactions can be distinguished on the basis of these IR spectra, leading to the formation of a new CN bond and to the decomposition of sugar skeletal bonds. The new IR bands suggest that the latter reaction results in the formation of new bonds, which are related to new polyether moieties. These results highlight the often ignored non-specific chemical reactions that take place between sugars and proteins, and demonstrate that the simultaneous application of FTIR and NMR spectroscopic analyses can detect and further characterize these types of sugar-protein interactions.
Topics: Arabinose; Cold Temperature; Glucose; Humans; Maillard Reaction; Nuclear Magnetic Resonance, Biomolecular; Proteins; Spectroscopy, Fourier Transform Infrared; Sucrose; Sugars; Xylose
PubMed: 31255896
DOI: 10.1016/j.saa.2019.02.085 -
Genes and Environment : the Official... Nov 2022Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA...
BACKGROUND
Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized.
RESULTS
In this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals.
CONCLUSION
Using this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained.
PubMed: 36380394
DOI: 10.1186/s41021-022-00254-1 -
Journal of the Science of Food and... Jul 2023The nixtamalization process improves the nutritional and technological properties of maize. This process generates nixtamalized maize bran as a by-product, which is a...
BACKGROUND
The nixtamalization process improves the nutritional and technological properties of maize. This process generates nixtamalized maize bran as a by-product, which is a source of arabinoxylans (AX). AX are polysaccharides constituted of a xylose backbone with mono- or di-arabinose substitutions, which can be ester-linked to ferulic acid (FA). The present study investigated the fine structural features and antioxidant capacity (AC) of nixtamalized maize bran arabinoxylans (MBAX) to comprehend the structure-radical scavenging capacity relationship in this polysaccharide deeply.
RESULTS
MBAX presented a molecular weight, intrinsic viscosity, and hydrodynamic radius of 674 kDa, 1.8 dL g , and 24.6 nm, respectively. The arabinose-to-xylose ratio (A/X) and FA content were 0.74 and 0.25 g kg polysaccharide, respectively. MBAX contained dimers (di-FA) and trimer (tri-FA) of FA (0.14 and 0.07 g kg polysaccharide, respectively). The main di-FA isomer was the 8-5' structure (80%). Fourier transform infrared spectroscopy confirmed MBAX molecular identity, and the second derivate of the spectral data revealed a band at 958 cm related to the presence of arabinose disubstitution. H-Nuclear magnetic resonance spectroscopy showed mono- and di-arabinose substitution in the xylan backbone with more monosubstituted residues. MBAX registered an AC of 25 and 20 μmol Trolox equivalents g polysaccharide despite a low FA content, using ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) and DPPH (1,1-diphenyl-2-picrylhydrazyl) methods, respectively.
CONCLUSION
AC in MBAX could be related to the high A/X ratio (mainly monosubstitution) and the high 8-5' di-FA proportion in this polysaccharide. © 2023 Society of Chemical Industry.
Topics: Xylans; Antioxidants; Zea mays; Xylose; Arabinose; Polysaccharides
PubMed: 36852427
DOI: 10.1002/jsfa.12531 -
Polymers Aug 2022Two polysaccharides from L. (CTLP-1 and CTLP-2) were purified, and their structures were analyzed by physical and chemical testing. CTLP-1 had a mass of 5900 Da that...
Two polysaccharides from L. (CTLP-1 and CTLP-2) were purified, and their structures were analyzed by physical and chemical testing. CTLP-1 had a mass of 5900 Da that was composed of arabinose, glucose, and galactose with a mass molar ratio of 6.7:4.2:1. The backbone of CTLP-1 was →1)-α-GalAp-(1→4)-α-Arap-(1→2)-α-Glup-(4→. CTLP-2 had a mass of 8200 Da that was composed of arabinose, glucose, and galactose with a mass molar ratio of 16.76:4.28:1. The backbone of CTLP-2 was →1)-α-Galp-(2,6 →1)-α-Arap-(4,6 →1)-α-Glup-(3→. Both of them exhibited a high reducing power, hydroxyl radical scavenging activity, DPPH radical scavenging activity and ABTS radical scavenging activity, moderate Fe chelating activity and superoxide anion scavenging activity, implying that they might be potential antioxidants.
PubMed: 36080585
DOI: 10.3390/polym14173510 -
The Protein Journal Aug 2023The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase,...
The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius.
Topics: Escherichia coli; Molecular Docking Simulation; Phylogeny; Kinetics; Glycoside Hydrolases; Substrate Specificity
PubMed: 37119380
DOI: 10.1007/s10930-023-10117-5 -
Bioresource Technology May 2020Alpha-L-arabinofuranoside arabinofuranohydrolase (ARA), more commonly known as alpha-L-arabinofuranosidase (E.C. number 3.2.1.55), is a hydrolytic enzyme, catalyzing the... (Review)
Review
Alpha-L-arabinofuranoside arabinofuranohydrolase (ARA), more commonly known as alpha-L-arabinofuranosidase (E.C. number 3.2.1.55), is a hydrolytic enzyme, catalyzing the cleavage of alpha-L-arabinose by acting on the non-reducing ends of alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linked arabinoxylans and arabinogalactans. ARA functions as debranching enzyme removing arabinose substituents from arabinoxylan and arabinoxylooligomers, thereby, boosting the hydrolysis of arabinoxylan fraction of hemicellulose and improving bioconversion of lignocellulosic biomass. Previously, comprehensive information on this enzyme has not been reviewed thoroughly. Therefore, the main aim of this review is to highlight the important properties of this interesting enzyme, microorganisms used for its production, and enhanced production using genetic engineering approach. An account on synergism with other biomass hydrolyzing enzymes and various industrial applications of this enzyme has also been provided along with an outlook on further research and development.
Topics: Arabinose; Biomass; Glycoside Hydrolases; Hydrolysis; Substrate Specificity; Xylans
PubMed: 32089440
DOI: 10.1016/j.biortech.2020.123019 -
Astrobiology May 2021Preferential uptake of either levorotatory (L) or dextrorotatory (D) enantiomer of a chiral molecule is a potential planetary life-detection method. On Earth, bacteria,...
Preferential uptake of either levorotatory (L) or dextrorotatory (D) enantiomer of a chiral molecule is a potential planetary life-detection method. On Earth, bacteria, as a rule, metabolize D-sugars and L-amino acids. Here, we use growth experiments to identify exceptions to the rule and their potential impact on the method's reliability. Our experiments involve six strains of and collective uptake of the sugars glucose and arabinose, and the amino acids alanine, glutamic acid, leucine, cysteine, and serine-all of which are highly soluble. We find that selective uptake is not evident unless (1) each sugar is tested individually and (2) multiple amino acids are tested together in a mixture. Combining sugars should be avoided because, as we show in bacteria, the same organisms may catabolize one sugar, glucose, in D-form and another sugar, arabinose, in L-form. Single amino acids should be avoided because bacteria can access certain proteinogenically incompatible enantiomers using specific racemases. Specifically, bacteria contain an alanine acid racemase and can catabolize D-alanine if no other D-amino acids are present. The proposed improvements would reliably separate nonselective chemical reactions from biological reactions and, if life is indicated, inform whether the selective patterns for amino acids and sugars are the same as on Earth.
Topics: Alanine; Amino Acids; Carbohydrates; Reproducibility of Results; Stereoisomerism
PubMed: 33885325
DOI: 10.1089/ast.2020.2381 -
Food & Function Mar 2021l-Arabinose is a kind of plant-specific five-carbon aldose with benefits in type 2 diabetes mellitus. It has been shown to have good properties in improving glucose...
l-Arabinose is a kind of plant-specific five-carbon aldose with benefits in type 2 diabetes mellitus. It has been shown to have good properties in improving glucose homeostasis, but the underlying molecular mechanisms are still not clear. Hepatic gluconeogenesis is critical for regulating glucose homeostasis. Here, this study aimed to investigate whether l-arabinose could improve glucose metabolism via suppressing hepatic gluconeogenesis. High-fat-high-sucrose diet (HFHSD) or high-sucrose diet (HSD)-fed mice were supplemented with or without l-arabinose for 12 weeks. Fasting blood glucose levels were measured and glucose tolerance test and the histological analysis were performed after l-arabinose administration. AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), peroxisome proliferator activated receptor-γ coactivator-1α (PGC1α), Forkhead box O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression levels were determined by RT-PCR and western blotting. As expected, l-arabinose apparently decreased body weight and attenuated hyperglycemia and glucose intolerance caused by HFHSD or HSD. l-Arabinose also had beneficial effects on glycogen synthesis by inactivating GSK3β. The expression levels of gluconeogenic genes were all decreased by l-arabinose administration in vivo and in vitro. In addition, our work revealed that AMPK is required for the inhibitory effects of l-arabinose on hepatic gluconeogenesis. l-Arabinose significantly up-regulated the phosphorylated levels of AMPK and its downstream protein ACC. Furthermore, blocking AMPK signaling through an inhibitor (compound C) or siAMPK significantly attenuated the inhibition of hepatic gluconeogenesis and the promotion of glycogen synthesis with l-arabinose, indicating that the inhibitory effect of l-arabinose on hepatic gluconeogenesis was AMPK dependent. Our work revealed that l-arabinose is a promising natural product for the regulation of hyperglycemia through inhibition of hepatic gluconeogenesis by activating AMPK.
Topics: AMP-Activated Protein Kinases; Animals; Arabinose; Blood Glucose; Disease Models, Animal; Gluconeogenesis; Hyperglycemia; Male; Mice; Mice, Inbred C57BL
PubMed: 33502423
DOI: 10.1039/d0fo02163f