scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

Features of the cellodextrinase gene from Fibrobacter succinogenes S85

1994 ◽  
Vol 40 (7) ◽  
pp. 592-596 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Gel Filtration ◽  
Fibrobacter Succinogenes ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Base Pairs ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Promoter Sequences ◽  
Putative Ribosome Binding Site ◽  
Coli Promoter

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.

scholarly journals Molecular-scale features that govern the effects of O-glycosylation on a carbohydrate-binding module

2015 ◽  
Vol 6 (12) ◽  
pp. 7185-7189 ◽  
Author(s):  
Xiaoyang Guan ◽  
Patrick K. Chaffey ◽  
Chen Zeng ◽  
Eric R. Greene ◽  
Liqun Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glycosylation Site ◽  
Glycan Structure ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosidic Linkage ◽  
Molecular Scale

The importance of the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage in controlling the effects of CBMO-glycosylation is shown.

scholarly journals Carbohydrate-Binding Module and Linker Allow Cold Adaptation and Salt Tolerance of Maltopentaose-Forming Amylase From Marine Bacterium Saccharophagus degradans 2-40T

2021 ◽  
Vol 12 ◽  
Author(s):  
Ning Ding ◽  
Boyang Zhao ◽  
Xiaofeng Ban ◽  
Caiming Li ◽  
B. V. Venkataram Prasad ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
Cold Adaptation ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Maximum Activity ◽  
Significant Loss ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Saccharophagus Degradans ◽  
Unfavorable Condition

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

scholarly journals Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

2005 ◽  
Vol 388 (3) ◽  
pp. 949-957 ◽  
Author(s):  
Masashi KIYOHARA ◽  
Keishi SAKAGUCHI ◽  
Kuniko YAMAGUCHI ◽  
Toshiyoshi ARAKI ◽  
Takashi NAKAMURA ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Carbohydrate Binding Modules ◽  
Hydrolysis Of ◽  
Hydrolase Family

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24

2009 ◽  
Vol 55 (9) ◽  
pp. 1113-1118 ◽  
Author(s):  
Gaofei Duan ◽  
Feng Han ◽  
Wengong Yu
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Alginate Lyase ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
His Tag ◽  
Consensus Region ◽  
Encoding Gene ◽  
Alginate Lyases ◽  
Pcr Techniques

The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 °C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0–10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+ can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of ~30 kDa, was found in AlyPI, which belongs to the ~60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.

Sequence Analysis of Potato α-Amylase Gene amyA2

2013 ◽  
Vol 643 ◽  
pp. 56-59 ◽  
Author(s):  
Yong Gang Wang ◽  
Jian Zhong Ma ◽  
Xue Qing Ma ◽  
Jin Ge Liu ◽  
Ming Jun Yang
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Amino Acid Level ◽  
Chemical Properties ◽  
Amino Acid Residues ◽  
Accession Number ◽  
Amylase Gene ◽  
Reading Frame ◽  
Beta Barrel ◽  
Physical And Chemical

The sequences of Potato α-Amylase Gene amyA2 was analysised by Bioinformatics, including its codon usage bias, physical and chemical properties, subcellular localization, and conserved structures. The results showed that the cDNA had a 1218 bp open reading frame and was referred to as amyA2, which encodes for an α-amylase with 405 amino acid residues (GenBank accession number: GU134783), and shared 98% identity with a published potato α-amylase (GenBank accession number: M79328.1) at the amino acid level. The amino sequences contains a catalytic domain (PF00128、SM00624) between 20 to 348 and a C-terminal beta-sheet domain between 349-407, which are similar to ones of the amylase family 13. Eight-stranded alpha/beta barrel was also found in the enzyme, which was thought as an active site of α-amylase.

scholarly journals Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE

1999 ◽  
Vol 181 (10) ◽  
pp. 3270-3276 ◽  
Author(s):  
Yutaka Tamaru ◽  
Roy H. Doi
Keyword(s):  
Surface Layer ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Caulobacter Crescentus ◽  
Scaffolding Protein ◽  
Glycosyl Hydrolases ◽  
Clostridium Cellulovorans ◽  
Reading Frame ◽  
Enzymatic Function ◽  
C Terminus

ABSTRACT The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovoranscellulosome, has been isolated and sequenced. engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus. engE is located downstream of an ORF, ORF1, that is homologous to theBacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans.

scholarly journals Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

1999 ◽  
Vol 65 (9) ◽  
pp. 4099-4107 ◽  
Author(s):  
Anne E. Cazemier ◽  
Jan C. Verdoes ◽  
Albert J. J. van Ooyen ◽  
Huub J. M. Op den Camp
Keyword(s):  
Amino Acids ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Cellulolytic Bacterium ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Encoding Genes

ABSTRACT Two xylanase-encoding genes, named xyn11A andxyn10B, were isolated from a genomic library ofCellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the twoCellulomonas species were identified.

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