scholarly journals Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

2021 ◽  
Vol 22 (23) ◽  
pp. 12822
Author(s):  
Sung Kyum Kim ◽  
Jong Eun Park ◽  
Jong Min Oh ◽  
Hoon Kim
Keyword(s):  
Amino Acids ◽  
Main Product ◽  
Glycosyl Hydrolase ◽  
Glycosyl Hydrolase Family ◽  
Chitinolytic Bacteria ◽  
Binding Domains ◽  
Alkaline Conditions ◽  
Molecular Masses ◽  
Hydrolase Family

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N′-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

scholarly journals Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

2008 ◽  
Vol 75 (4) ◽  
pp. 1135-1143 ◽  
Author(s):  
Karina Pokusaeva ◽  
Mary O'Connell-Motherway ◽  
Aldert Zomer ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
Glycosyl Hydrolase ◽  
Hydrolytic Activity ◽  
Glycosyl Hydrolase Family ◽  
Bifidobacterium Breve ◽  
Transglycosylation Activity ◽  
Encoding Genes ◽  
Temperature Optima ◽  
Hydrolase Family

ABSTRACT Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His12 sequence was incorporated (Agl1His and Agl2His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1His exhibits higher thermo and pH optima than Agl2His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.

scholarly journals Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils

2003 ◽  
Vol 2 (5) ◽  
pp. 1091-1098 ◽  
Author(s):  
Li Li ◽  
Jürgen Fröhlich ◽  
Peter Pfeiffer ◽  
Helmut König
Keyword(s):  
Glycosyl Hydrolase ◽  
Gut Contents ◽  
Cellulolytic Enzymes ◽  
Glycosyl Hydrolase Family ◽  
Mrna Pool ◽  
Termite Gut ◽  
Significant Homology ◽  
Molecular Masses ◽  
Gut Symbionts ◽  
Hydrolase Family

ABSTRACT Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.

scholarly journals Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

2002 ◽  
Vol 277 (46) ◽  
pp. 44035-44043 ◽  
Author(s):  
Tariq A. Tahir ◽  
Jean-Guy Berrin ◽  
Ruth Flatman ◽  
Alain Roussel ◽  
Peter Roepstorff ◽  
...  
Keyword(s):  
Binding Sites ◽  
Glycosyl Hydrolase ◽  
Glycosyl Hydrolase Family ◽  
Inhibitor Binding ◽  
Hydrolase Family

scholarly journals A stress-induced rice (Oryza sativa L.) β-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

2007 ◽  
Vol 408 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Rodjana Opassiri ◽  
Busarakum Pomthong ◽  
Takashi Akiyama ◽  
Massalin Nakphaichit ◽  
Tassanee Onkoksoong ◽  
...  
Keyword(s):  
Amino Acids ◽  
Oryza Sativa ◽  
Oryza Sativa L ◽  
Glycosyl Hydrolase ◽  
Catalytic Efficiency ◽  
Degree Of Polymerization ◽  
Mature Protein ◽  
Glycosyl Hydrolase Family ◽  
Submergence Stress ◽  
Hydrolase Family

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) β-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a β-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including β-D-glucoside, α-L-arabinoside, β-D-fucoside, β-D-galactoside, β-D-xyloside and β-D-cellobioside, as well as β-(1,4)-linked glucose oligosaccharides and β-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/Km) for hydrolysis of β-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-β-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.

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