scholarly journals Novel α-Glucosidase from Aspergillus nidulans with Strong Transglycosylation Activity

2002 ◽  
Vol 68 (3) ◽  
pp. 1250-1256 ◽  
Author(s):  
Naoki Kato ◽  
Sachie Suyama ◽  
Masao Shirokane ◽  
Masashi Kato ◽  
Tetsuo Kobayashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Glycosyl Hydrolase ◽  
Amino Acid Sequences ◽  
Glycosyl Hydrolase Family ◽  
Transglycosylation Activity ◽  
Single Polypeptide ◽  
Hydrolysis Of ◽  
Hydrolase Family

ABSTRACT Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.

scholarly journals Molecular Cloning of Endo-β-d-1,4-Glucanase Genes, rce1, rce2, and rce3, from Rhizopus oryzae

2003 ◽  
Vol 185 (5) ◽  
pp. 1749-1756 ◽  
Author(s):  
Tatsuki Moriya ◽  
Koichiro Murashima ◽  
Akitaka Nakane ◽  
Koji Yanai ◽  
Naomi Sumida ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Rhizopus Oryzae ◽  
Glycosyl Hydrolase ◽  
Amino Acid Sequences ◽  
Glycosyl Hydrolase Family ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Cellulose Binding ◽  
Hydrolase Family

ABSTRACT Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.

scholarly journals A novel α-glucosidase from the acidophilic archaeon Ferroplasma acidiphilum strain Y with high transglycosylation activity and an unusual catalytic nucleophile

2005 ◽  
Vol 391 (2) ◽  
pp. 269-276 ◽  
Author(s):  
Manuel Ferrer ◽  
Olga V. Golyshina ◽  
Francisco J. Plou ◽  
Kenneth N. Timmis ◽  
Peter N. Golyshin
Keyword(s):  
Glycosyl Hydrolase ◽  
Soluble Starch ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Preference Order ◽  
Glycosyl Hydrolase Family ◽  
Significant Similarity ◽  
Transglycosylation Activity ◽  
Membrane Bound ◽  
Hydrolase Family

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound α-glucosidase (αGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an unusual catalytic site consisting of a threonine and a histidine residue. The highest α-glucosidase activity was found at low pH, 2.4–3.5, and the substrate preference order was: sucrose>maltose>maltotriose ≫maltotetraose≫malto-oligosaccharides from maltopentaose to maltoheptaose⋙soluble starch (kcat/Km was 293.0, 197.0, 18.8, 0.3 and 0.02 s−1·mM−1 respectively). The enzyme was able to transfer glucosyl groups from maltose as donor, to produce exclusively maltotriose (up to 300 g/l). Chemical modification and electrospray ionization MS analysis of 5-fluoro-α-D-glucopyranosyl-enzyme derivatives, coupled with site-directed mutagenesis, strongly suggested that the putative catalytic nucleophile in this enzyme is Thr212. Iron was found to be essential for enzyme activity and integrity, and His390 was shown to be essential for iron binding. These results suggest that the metalloenzyme αGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.

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 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.

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 Distinct Glycosyl Hydrolase Family 78 α-l-Rhamnosidases from Pediococcus acidilactici

2011 ◽  
Vol 77 (18) ◽  
pp. 6524-6530 ◽  
Author(s):  
Herbert Michlmayr ◽  
Walter Brandes ◽  
Reinhard Eder ◽  
Christina Schümann ◽  
Andrés M. del Hierro ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Pediococcus Acidilactici ◽  
Glycosyl Hydrolase Family ◽  
Negative Effects ◽  
Content Type ◽  
Linalool Oxide ◽  
Hydrolysis Of ◽  
Flavanone Glycoside ◽  
Hydrolase Family

ABSTRACTα-l-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ramandram2) fromPediococcus acidilacticiDSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool andcis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substratep-nitrophenyl-α-l-rhamnopyranoside (Vmax= 243 U mg−1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax= 34 U mg−1) and, especially, rutinose (Vmax= 1,200 U mg−1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.

scholarly journals New Chitosan-Degrading Strains That Produce Chitosanases Similar to ChoA of Mitsuaria chitosanitabida

2005 ◽  
Vol 71 (9) ◽  
pp. 5138-5144 ◽  
Author(s):  
ChoongSoo Yun ◽  
Daiki Amakata ◽  
Yasuhiro Matsuo ◽  
Hideyuki Matsuda ◽  
Makoto Kawamukai
Keyword(s):  
Southern Hybridization ◽  
Glycosyl Hydrolase ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Glycosyl Hydrolase Family ◽  
Broad Variety ◽  
Hydrolase Family ◽  
The 16S Rrna Gene ◽  
Sequence Similarities

ABSTRACT The betaproteobacterium Mitsuaria chitosanitabida (formerly Matsuebacter chitosanotabidus) 3001 produces a chitosanase (ChoA) that is classified in glycosyl hydrolase family 80. While many chitosanase genes have been isolated from various bacteria to date, they show limited homology to the M. chitosanitabida 3001 chitosanase gene (choA). To investigate the phylogenetic distribution of chitosanases analogous to ChoA in nature, we identified 67 chitosan-degrading strains by screening and investigated their physiological and biological characteristics. We then searched for similarities to ChoA by Western blotting and Southern hybridization and selected 11 strains whose chitosanases showed the most similarity to ChoA. PCR amplification and sequencing of the chitosanase genes from these strains revealed high deduced amino acid sequence similarities to ChoA ranging from 77% to 99%. Analysis of the 16S rRNA gene sequences of the 11 selected strains indicated that they are widely distributed in the β and γ subclasses of Proteobacteria and the Flavobacterium group. These observations suggest that the ChoA-like chitosanases that belong to family 80 occur widely in a broad variety of bacteria.

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