scholarly journals Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

2015 ◽  
Vol 81 (19) ◽  
pp. 6610-6620 ◽  
Author(s):  
Vincent Valk ◽  
Wieger Eeuwema ◽  
Fean D. Sarian ◽  
Rachel M. van der Kaaij ◽  
Lubbert Dijkhuizen
Keyword(s):  
Enzyme System ◽  
Recombinant Expression ◽  
Potato Starch ◽  
Carbohydrate Binding ◽  
Starch Granules ◽  
Catalytic Core ◽  
Content Type ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTThe bacteriumMicrobacterium aurumstrain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomainM. aurumB8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded thatM. aurumB8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.

scholarly journals Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

2012 ◽  
Vol 78 (17) ◽  
pp. 6161-6171 ◽  
Author(s):  
Christoph Sygmund ◽  
Daniel Kracher ◽  
Stefan Scheiblbrandner ◽  
Kawah Zahma ◽  
Alfons K. G. Felice ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Enzyme System ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Carbohydrate Binding ◽  
Ph Optimum ◽  
Content Type ◽  
Polysaccharide Monooxygenase

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

scholarly journals Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (19) ◽  
pp. 7048-7059 ◽  
Author(s):  
Libin Ye ◽  
Xiaoyun Su ◽  
George E. Schmitz ◽  
Young Hwan Moon ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Cellulose Hydrolysis ◽  
Thermophilic Bacterium ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
C Terminus ◽  
Carbohydrate Binding Modules ◽  
Β Sheets ◽  
Biochemical Analyses

ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.

scholarly journals Novel Family of Carbohydrate-Binding Modules Revealed by the Genome Sequence of Spirochaeta thermophila DSM 6192

2011 ◽  
Vol 77 (15) ◽  
pp. 5483-5489 ◽  
Author(s):  
Angel Angelov ◽  
Christoph Loderer ◽  
Susanne Pompei ◽  
Wolfgang Liebl
Keyword(s):  
Genome Sequence ◽  
Sequence Data ◽  
Cellulose Degradation ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Carbohydrate Binding ◽  
Functional Screening ◽  
Content Type ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTSpirochaeta thermophilais a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on theS. thermophilagenome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from theS. thermophilaDSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected inSpirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes ofCytophaga fermentansandMahella australiensis, both of which are phylogenetically very distant fromS. thermophilaand noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

scholarly journals Exposing the Secrets of Two Well-Known Lactobacillus casei Phages, J-1 and PL-1, by Genomic and Structural Analysis

2014 ◽  
Vol 80 (22) ◽  
pp. 7107-7121 ◽  
Author(s):  
Maria Eugenia Dieterle ◽  
Charles Bowman ◽  
Carlos Batthyany ◽  
Esteban Lanzarotti ◽  
Adrián Turjanski ◽  
...  
Keyword(s):  
Lactobacillus Casei ◽  
Sequence Similarity ◽  
Proteolytic Processing ◽  
Mass Spectrometry Analysis ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Related Phage ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACTBacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult usingLactobacillus caseistrain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in otherSiphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically toLactobacillus casei/paracaseicells, and the addition ofl-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls ofL. caseiATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into howLactobacillusphages interact with their hosts at the first steps of infection.

scholarly journals Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Junyan Ma ◽  
Qian Li ◽  
Haidong Tan ◽  
Hao Jiang ◽  
Kuikui Li ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Jerusalem Artichoke ◽  
Fine Tuning ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Secondary Structure Analysis ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Carbohydrate Binding Modules

Abstract Background Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. Results Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. Conclusion The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

scholarly journals Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization

2003 ◽  
Vol 372 (3) ◽  
pp. 905-910 ◽  
Author(s):  
Tzur PALDI ◽  
Ilan LEVY ◽  
Oded SHOSEYOV
Keyword(s):  
Aspergillus Niger ◽  
Corn Starch ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Carbohydrate Binding ◽  
Amylolytic Enzymes ◽  
Binding Domains ◽  
C Terminus ◽  
Carbohydrate Binding Modules ◽  
Modified Starches

Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95–96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch.

scholarly journals Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

2015 ◽  
Vol 81 (6) ◽  
pp. 2006-2014 ◽  
Author(s):  
Dong-Dong Meng ◽  
Yu Ying ◽  
Xiao-Hua Chen ◽  
Ming Lu ◽  
Kang Ning ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Glycoside Hydrolase ◽  
Residual Activity ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Evolutionary Strategy ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Carbohydrate Binding Modules ◽  
Interdomain Interactions

ABSTRACTXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified fromCaldicellulosiruptorsp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed inEscherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol−1versus 94.7 U nmol−1) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.

Sign in / Sign up

Export Citation Format

Share Document