Cloning of a xylanase gene xyn2A from rumen fungus Neocallimastix sp. GMLF2 in Escherichia coli and its partial characterization

Biologia ◽  
2009 ◽  
Vol 64 (4) ◽  
Author(s):  
Ismail Akyol ◽  
Ugur Comlekcioglu ◽  
Bulent Kar ◽  
M. Ekinci ◽  
Emin Ozkose
Keyword(s):  
Escherichia Coli ◽  
Xylanase Activity ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Gene Coding ◽  
The Family ◽  
Rumen Fungus ◽  
Polymerase Chain ◽  
Encoding Gene ◽  
Hydrolase Family

AbstractAnaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.

scholarly journals A New β-Galactosidase from the Antarctic Bacterium Alteromonas sp. ANT48 and Its Potential in Formation of Prebiotic Galacto-Oligosaccharides

Marine Drugs ◽  
2019 ◽  
Vol 17 (11) ◽  
pp. 599 ◽  
Author(s):  
Li ◽  
Zhu ◽  
Xing
Keyword(s):  
Escherichia Coli ◽  
Molecular Docking ◽  
Glycoside Hydrolase ◽  
Intestinal Flora ◽  
Glycoside Hydrolase Family ◽  
Initial Activity ◽  
Antarctic Bacterium ◽  
Encoding Gene ◽  
The Antarctic ◽  
Hydrolase Family

As an important medical enzyme, β-galactosidases catalyze transgalactosylation to form prebiotic Galacto-Oligosaccharides (GOS) that assist in improving the effect of intestinal flora on human health. In this study, a new glycoside hydrolase family 2 (GH2) β-galactosidase-encoding gene, galA, was cloned from the Antarctic bacterium Alteromonas sp. ANT48 and expressed in Escherichia coli. The recombinant β-galactosidase GalA was optimal at pH 7.0 and stable at pH 6.6–7.0, which are conditions suitable for the dairy environment. Meanwhile, GalA showed most activity at 50 °C and retained more than 80% of its initial activity below 40 °C, which makes this enzyme stable in normal conditions. Molecular docking with lactose suggested that GalA could efficiently recognize and catalyze lactose substrates. Furthermore, GalA efficiently catalyzed lactose degradation and transgalactosylation of GOS in milk. A total of 90.6% of the lactose in milk could be hydrolyzed within 15 min at 40 °C, and the GOS yield reached 30.9%. These properties make GalA a good candidate for further applications.

scholarly journals Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuya Kuritani ◽  
Kohei Sato ◽  
Hideo Dohra ◽  
Seiichiro Umemura ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metabolic Pathway ◽  
Glycoside Hydrolase ◽  
Gene Locus ◽  
Glycoside Hydrolase Family ◽  
Sequential Reaction ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Hydrolase Family

AbstractLevoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

scholarly journals Overexpression of Lipase Gene from Alcaligenes sp. JG3 and its Activity toward Hydrolysis Reaction

2019 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
Norman Yoshi Haryono ◽  
Winarto Haryadi ◽  
Tri Joko Raharjo
Keyword(s):  
Specific Activity ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Lipase Gene ◽  
High Quality ◽  
Atp Binding Site ◽  
E Coli ◽  
Transporter Protein ◽  
Encoding Gene ◽  
Hydrolase Family

Bacterial lipase holds an important role as a new source for many industrial catalysts. The investigation and understanding of the lipase-encoding gene become apparent as the key step for generating high-quality lipase as biocatalyst for many chemical reactions. In this study, bacterial lipase from Alcaligenes sp. JG3 was produced via overexpression gene method. This specific lipase was successfully overexpressed using pQE-30 vector and E. coli M15[pREP4] as host, producing His-tagged protein sized 46 kDa and was able to hydrolyze triacylglycerol from olive oil with the calculated unit activity and specific activity of 0.012 U and 1.175 U/mg respectively. The in silico investigation towards lipase JG3 revealed that it was categorized as ABC transporter protein as opposed to the conventional hydrolase family. Lastly, amino acid sequences SGSGKTT from lipase JG3 was highly conserved sequences and was predicted as the ATP-binding site but the catalytic triad of serine, histidine, and aspartate has not been solved yet.

scholarly journals Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11

2002 ◽  
Vol 184 (15) ◽  
pp. 4124-4133 ◽  
Author(s):  
Kaveh Emami ◽  
Tibor Nagy ◽  
Carlos M. G. A. Fontes ◽  
Luis M. A. Ferreira ◽  
Harry J. Gilbert
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Xylanase Activity ◽  
Glycoside Hydrolase Family ◽  
Side Chains ◽  
Temporal Regulation ◽  
Degrading Bacterium ◽  
Genes Encoding ◽  
Combined Action ◽  
Hydrolase Family

ABSTRACT Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

scholarly journals A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

scholarly journals Characterization and Functional Importance of Two Glycoside Hydrolase Family 16 Genes from the Rice White Tip Nematode Aphelenchoides besseyi

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 374
Author(s):  
Hui Feng ◽  
Dongmei Zhou ◽  
Paul Daly ◽  
Xiaoyu Wang ◽  
Lihui Wei
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Parasitic Nematodes ◽  
Glycoside Hydrolase Family ◽  
Plant Parasitic Nematodes ◽  
Aphelenchoides Besseyi ◽  
Rice White Tip Nematode ◽  
Hydrolase Family ◽  
Plant Parasitic

The glycoside hydrolase family 16 (GH16) is widely found in prokaryotes and eukaryotes, and hydrolyzes the β-1,3(4)-linkages in polysaccharides. Notably, the rice white tip nematode Aphelenchoides besseyi harbors a higher number of GH16s compared with other plant-parasitic nematodes. In this work, two GH16 genes, namely AbGH16-1 and AbGH16-2, were isolated and characterized from A. besseyi. The deduced amino acid sequences of AbGH16-1 and AbGH16-2 contained an N-terminal signal peptide and a fungal Lam16A glucanase domain. Phylogenetic analysis revealed that AbGH16-1 and AbGH16-2 clustered with ascomycete GH16s, suggesting AbGH16-1 and AbGH16-2 were acquired by horizontal gene transfer from fungi. In situ hybridization showed that both AbGH16-1 and AbGH16-2 were specifically expressed in the nematode gonads, correlating with qPCR analysis that showed the high transcript levels of the two genes in the female nematodes. AbGH16-1 and AbGH16-2 were also significantly induced in nematodes feeding on Botrytis cinerea. Characterization of the recombinant protein showed AbGH16-1 and AbGH16-2 displayed pronounced inhibition of both conidial germination and germ tube elongation of B. cinerea. In addition, silencing of AbGH16-1 and AbGH16-2 by RNA interference significantly decreased the reproduction ability of A. besseyi and had a profound impact on the development process of offspring in this nematode. These findings have firstly proved that GH16s may play important roles in A.besseyi feeding and reproduction on fungi, which thus provides novel insights into the function of GH16s in plant-parasitic nematodes.

scholarly journals Molecular Identification of Virulence Genes of Escherichia coli Isolated from Cow Milk and Its Products in Abuja, Nigeria

2020 ◽  
pp. 11-18
Author(s):  
E. C. Okechukwu ◽  
E. U. Amuta ◽  
G. M. Gberikon ◽  
N. Chima ◽  
B. Yakubu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Foodborne Pathogen ◽  
Cow Milk ◽  
E Coli ◽  
Milk Samples ◽  
The Family ◽  
High Prevalence ◽  
Polymerase Chain

Shiga toxin-producing Escherichia coli have been identified as an emerging foodborne pathogen which portends serious risk to human health. Cow milk and its products are potential sources of shiga toxin-producing Escherichia coli. A relatively small number from the family of shiga toxin-producing Escherichia coli are pathogenic. It becomes necessary that Cow milk and milk products are regularly screened for the presence of virulence genes in microbes. The study aimed to genetically determine the presence of virulence genes that are characteristic of Enterohaemorrhagic E. coli in 600 milk samples. The E. coli isolates were recovered from the milk samples (n=35), biochemically examined and genetically screened for virulence genes by multiplex Polymerase Chain Reaction (PCR). The results of the molecular profiling revealed that (stx2) was detected in 17(60.7%), (hlyA) 11(39.3%) and eae genes 8(28.6%) of the E. coli isolates respectively, while (stx1) was not detected. The results indicated a high prevalence of virulent shiga toxin-producing Escherichia coli in the milk samples. Priority attention should be given to this microbe as it will demand stringent steps in the detection given that they are known to be rigorous in identification.

scholarly journals One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

2021 ◽  
Author(s):  
Jiawen Liu ◽  
Cong Liu ◽  
Shilei Qiao ◽  
Zhen Dong ◽  
Di Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wheat Bran ◽  
Xylanase Activity ◽  
Maximal Activity ◽  
Fermentation Medium ◽  
Growth Conditions ◽  
Glycoside Hydrolase Family ◽  
E Coli ◽  
Response Surface Optimization ◽  
One Step

Abstract One-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of having cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombinant Escherichia coli through one-step fermentation was investigated here. A xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60°C and pH 8.0. Its activity retained more than 60% after incubation at 70°C for 4 hours, showing a good stability. The recombinant E. coli successfully secreted xylanases that directly hydrolyzed wheat bran to XOS in fermentation medium. The generated XOS consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The yield reached 5.3 mg/mL at 10% of wheat bran, which is higher than previous reports employing one-step fermentation. Nitrogen source type could also affect XOS yield by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied by response surface optimization. The maximal yield emerged at 44.3°C, pH 7.98, which is affected by characteristics of the xylanase and growth conditions of E. coli. This work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and yield for production of XOS.

scholarly journals Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119

1991 ◽  
Vol 280 (1) ◽  
pp. 187-191 ◽  
Author(s):  
M F Fillat ◽  
W E Borrias ◽  
P J Weisbeek
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Recombinant Protein ◽  
Expression Vector ◽  
Transcriptional Level ◽  
Wild Type ◽  
Chain Reaction ◽  
Gene Coding ◽  
Polymerase Chain ◽  
High Concentrations

The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR). The gene is transcribed into a 1250-base transcript. The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron. The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli. High concentrations of flavodoxin were found (20% of total protein). The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin.

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