scholarly journals Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 680
Author(s):  
Do Young Kim ◽  
Jonghoon Kim ◽  
Yung Mi Lee ◽  
Jong Suk Lee ◽  
Dong-Ha Shin ◽  
...  
Keyword(s):  
Tween 80 ◽  
Maximum Activity ◽  
Soil Bacterium ◽  
Glycoside Hydrolase Family ◽  
Antarctic Soil ◽  
Birchwood Xylan ◽  
Uncultured Bacterium ◽  
Cold Adapted ◽  
Beechwood Xylan ◽  
Key Enzyme

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

scholarly journals Screening of Novel Laccase Producers—Isolation and Characterization of Cold-Adapted Laccase from Kabatiella bupleuri G3 Capable of Synthetic Dye Decolorization

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 828
Author(s):  
Katarzyna M. Wiśniewska ◽  
Aleksandra Twarda-Clapa ◽  
Aneta M. Białkowska
Keyword(s):  
Morphological Characteristics ◽  
Tween 80 ◽  
Dye Decolorization ◽  
Defined Medium ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Its2 Region ◽  
Synthetic Dyes ◽  
Cold Adapted ◽  
Isolation And Characterization

Psychrophilic laccases catalyzing the bond formation in mild, environmentally friendly conditions are one of the biocatalysts at the focus of green chemistry. Screening of 41 cold-adapted strains of yeast and yeast-like fungi revealed a new laccase-producing strain, which was identified as Kabatiella bupleuri G3 IBMiP according to the morphological characteristics and analysis of sequences of the D1/D2 regions of 26S rDNA domain and the ITS1–5,8S–ITS2 region. The extracellular activity of laccase in reaction with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at the optimal pH 3.5 was 215 U/L after 15 days of growth in a medium with waste material and 126 U/L after 25 days of cultivation in a defined medium. Copper (II) ions (0.4 mM), Tween 80 (1.0 mM) and ascorbic acid (5.0 mM) increased the production of laccase. The optimum temperature for enzyme operation is in the range of 30–40 °C and retains over 60% of the maximum activity at 10 °C. New laccase shows high thermolability—half-life at 40 °C was only 60 min. Enzyme degradation of synthetic dyes was the highest for crystal violet, i.e., 48.6% after 1-h reaction with ABTS as a mediator. Outcomes of this study present the K. bupleuri laccase as a potential psychrozyme for environmental and industrial applications.

scholarly journals Cloning, Expression and Characterization of a Novel Cold-adapted β-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 469 ◽  
Author(s):  
Jingjing Sun ◽  
Congyu Yao ◽  
Wei Wang ◽  
Zhiwei Zhuang ◽  
Junzhong Liu ◽  
...  
Keyword(s):  
Deep Sea ◽  
Glycoside Hydrolase ◽  
Sea Water ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Cold Adapted ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Substrate Hydrolysis

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S−1 at 35 °C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

Temperature sensitivity of Antarctic soil‐humic substance degradation by cold‐adapted bacteria

2021 ◽  
Author(s):  
Dockyu Kim ◽  
Ha Ju Park ◽  
Mincheol Kim ◽  
Seulah Lee ◽  
Soon Gyu Hong ◽  
...  
Keyword(s):  
Humic Substance ◽  
Temperature Sensitivity ◽  
Antarctic Soil ◽  
Cold Adapted

scholarly journals The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

2021 ◽  
Author(s):  
Matthew P. Ostrowski ◽  
Sabina Leanti La Rosa ◽  
Benoit J. Kunath ◽  
Andrew Robertson ◽  
Gabriel Pereira ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Xanthan Gum ◽  
Enrichment Culture ◽  
Food Additives ◽  
Food Additive ◽  
Glycoside Hydrolase Family ◽  
Human Gut ◽  
High Molecular Weight Polymer ◽  
Molecular Weight Polymer ◽  
Uncultured Bacterium

SummaryThe diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

scholarly journals Characterization of a novel 3-O-α-D-galactosyl-α-L-arabinofuranosidase for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum

2021 ◽  
Author(s):  
Yuki Sasaki ◽  
Ayako Horigome ◽  
Toshitaka Odamaki ◽  
Jin-Zhong Xiao ◽  
Akihiro Ishiwata ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Bifidobacterium Longum ◽  
Gum Arabic ◽  
Glycoside Hydrolase Family ◽  
Type Ii ◽  
Cooperative Action ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Degradative Enzymes

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum. Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052, and classified it into the glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP and β-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of β-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum. Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the non-assimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type-II AG degradative enzymes in B. longum. The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longum. IMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase-carrier strains may affect GAfase-noncarrier strains that express other type-II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

scholarly journals Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

2021 ◽  
Author(s):  
Gregory S Bulmer ◽  
Fang Wei Yuen ◽  
Naimah Begum ◽  
Bethan S Jones ◽  
Sabine S Flitsch ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Enzyme Specificity ◽  
Fungal Enzymes ◽  
Glycoside Hydrolase Family 43 ◽  
Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

scholarly journals Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 174 ◽  
Author(s):  
Manami Kobayashi ◽  
Yuya Kumagai ◽  
Yohei Yamamoto ◽  
Hajime Yasui ◽  
Hideki Kishimura
Keyword(s):  
Main Product ◽  
Specific Activity ◽  
Red Alga ◽  
Glycoside Hydrolase Family ◽  
Bifidobacterium Adolescentis ◽  
Glycoside Hydrolase Family 43 ◽  
Key Enzyme ◽  
Hydrolysis Of ◽  
Low Activity ◽  
Hydrolase Family

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

scholarly journals Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

Biology ◽  
2013 ◽  
Vol 2 (1) ◽  
pp. 177-188 ◽  
Author(s):  
Renaud Berlemont ◽  
Olivier Jacquin ◽  
Maud Delsaute ◽  
Marcello La Salla ◽  
Jacques Georis ◽  
...  
Keyword(s):  
Antarctic Soil ◽  
Cold Adapted ◽  
Soil Metagenome

scholarly journals Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

2001 ◽  
Vol 183 (16) ◽  
pp. 4702-4708 ◽  
Author(s):  
Stéphane Benoit ◽  
James E. Posey ◽  
Matthew R. Chenoweth ◽  
Frank C. Gherardini
Keyword(s):  
Treponema Pallidum ◽  
Maximum Growth ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Phosphoglycerate Mutase ◽  
Heat Sensitivity ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
Key Enzyme

ABSTRACT In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP inT. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn2+ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.

A novel endo-β-1,4-xylanase GH30 from Dickeya dadantii DCE-01: Clone, expression, characterization, and ramie biological degumming function

2017 ◽  
Vol 89 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Ruijun Wang ◽  
Zhengchu Liu ◽  
Lifeng Cheng ◽  
Shengwen Duan ◽  
Xiangyuan Feng ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Ph Value ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolase Family ◽  
Dickeya Dadantii ◽  
Beechwood Xylan ◽  
Expression Characterization ◽  
Hydrolysis Of ◽  
Hydrolase Family

Xylanase plays an important role in the hydrolysis of hemicellulose and has gained much attention in the field of biological degumming. The research for xylanases with cellulase-free and high activity for biological degumming has intensified in recent years. In the present research, heterologous expression of a novel endo-β-1,4-xylanase (GH30) from Dickeya dadantii DCE-01 in Escherichia coli BL21 (DE3) was reported. Biochemical characterization of the enzyme and a potential application in ramie biological degumming was discussed. The results showed that the xylanase gene consists of 1251 nucleotides, belonging to glycoside hydrolase family 30 (GH30). The optimal activity of the xylanase was observed at 50℃ and a pH value of 6.4. The Km and Vmax values for beechwood xylan were 14.25 mg/mL and 296.6 μmol/mg, respectively. The catalytic activity was enhanced by addition of 1 mM Cu2+, Ca2+, Mg2+, and K+. The recombinant enzyme was specific for xylan substrates. The enzyme exhibited hydrolytic activity toward ramie hemicellulose. The recombinant xylanase could be effectively applied to ramie degumming.

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