scholarly journals A Single Mutation in the Carbohydrate-Binding Module Enhances Cellulase Activity in Bacillus Amyloliquefaciens Mutant

2021 ◽  
Vol 18 (18) ◽  
Author(s):  
Nitipol POLSA ◽  
Chomphunuch SONGSIRIRITTHIGUL ◽  
Wasana SUYOTHA ◽  
Sugunya SUEBSAN ◽  
Somboon ANUNTALABHOCHAI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Amyloliquefaciens ◽  
Cellulase Activity ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Single Mutation ◽  
Plasma Technology ◽  
Lignocellulosic Wastes ◽  
Cellulose Binding ◽  
Hydrolysis Activity

From our earlier work, we modified the carbohydrate-binding module (CBM) of Bacillus amyloliquefaciens to increase cellulase activity using cold plasma technology. The cellulase gene (BglC-M) from the mutant was expressed in Escherichia coli BL21(DE3) under the T7 promoter. The hydrolysis activity of the cellulase mutant (BglC-M) was approximately 2.5-fold higher than the control (BglC-W) over a wide range of pH and temperature conditions. The amino acid sequence of the mutant BglC-M contained 471 residues that were almost identical to the control BglC-W. Only a single amino acid, lysine, was replaced by glutamic acid at position 370 (K370E) within the carbohydrate-binding module (CBM). Structure prediction and substrate docking of BglC-M indicated that the single mutation (K370E) might involve cellulose binding of the β-sandwich facilitated by hydrogen bonding. The docking study of cellopentaose with the model structure of BglC-M indicated that the replacement of lysine-370 led to the formation of a hydrogen bond with 436Y, which has a shorter distance (2.6 Å) compared with the control (5.4 Å). As a result, the structure becomes more compact and stable, resulting in increased catalytic efficiency. Finally, the biomass hydrolysis ability of cellulase was investigated on lignocellulosic wastes such as pineapple peel, corncob, and durian peel. The BglC-M enzyme showed a more significant amount of reducing sugar released from all lignocellulosic wastes than the control. This was the first evidence that altering the base composition of the cellulose binding module enhanced the catalytic activity. HIGHLIGHTS Increasing cellulase activity of Bacillus amyloliquefaciens using plasma technology Mutation at cellulose-binding module enhance cellulase hydrolysis activity Greater cellulase activity in the hydrolysis on lignocellulosic wastes GRAPHICAL ABSTRACT

scholarly journals Oxidized Cellulose Binding to Allergens with a Carbohydrate-Binding Module Attenuates Allergic Reactions

2010 ◽  
Vol 186 (2) ◽  
pp. 1240-1247 ◽  
Author(s):  
Nir Shani ◽  
Ziv Shani ◽  
Oded Shoseyov ◽  
Rufayda Mruwat ◽  
David Shoseyov
Keyword(s):  
Carbohydrate Binding ◽  
Allergic Reactions ◽  
Carbohydrate Binding Module ◽  
Oxidized Cellulose ◽  
Cellulose Binding

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.

scholarly journals Extra tyrosine in the carbohydrate-binding module ofIrpex lacteusXyn10B enhances its cellulose-binding ability

2015 ◽  
Vol 79 (5) ◽  
pp. 738-746 ◽  
Author(s):  
Hiroto Nishijima ◽  
Kouichi Nozaki ◽  
Masahiro Mizuno ◽  
Tsutomu Arai ◽  
Yoshihiko Amano
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Ability ◽  
Cellulose Binding

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 Two pectate lyases from Caldicellulosiruptor bescii with the same CALG domain had distinct properties on plant biomass degradation

2020 ◽  
Author(s):  
Hamed I. Hamouda ◽  
Nasir Ali ◽  
Hang Su ◽  
Jie Feng ◽  
Ming Lu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plant Biomass ◽  
Degradation Products ◽  
Amino Acid Identity ◽  
Thermophilic Bacterium ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Acid Identity ◽  
Pectate Lyases ◽  
Caldicellulosiruptor Bescii

AbstractPectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that carry pectinolytic enzymes. Pectate lyases that cleave α-1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making and fruit softening. However, reports on pectate lyases with high thermostability are few. Two pectate lyases (CbPL3 and CbPL9) from a thermophilic bacterium Caldicellulosiruptor bescii were investigated. Although these two enzymes belonged to different families of polysaccharide lyase, both were Ca2+-dependent. Similar biochemical properties were shown under optimized conditions 80 °C–85 °C and pH 8–9. However, the degradation products on pectin and polygalacturonic acids (pGA) were different, revealing the distinct mode of action. A concanavalin A-like lectin/glucanase (CALG) domain, located in the N-terminus of two CbPLs, shares 100% amino acid identity. CALG-truncated mutant of CbPL9 showed lower activities than the wild-type, whereas the CbPL3 with CALG knock-out portion was reported with enhanced activities, thereby revealing the different roles of CALG in two CbPLs. I-TASSER predicted that the CALG in two CbPLs is structurally close to the family 66 carbohydrate binding module (CBM66). Furthermore, substrate-binding assay indicated that the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but CALG showed weak interaction with a number of lignocellulosic carbohydrates, except sodium carboxymethyl cellulose and sodium alginate. Finally, scanning electron microscope analysis and total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for degradation of plant biomass.ImportanceThermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium Caldicellulosiruptor bescii. The two enzymes had high optimum temperature, low optimum pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding module (CBM66) was identified in two CbPLs with sharing 100% amino acid identity. Deletion of CBM66 obviously decreased the activity of CbPL9, but increase the activity and thermostability of CbPL3, suggesting the different roles of CBM66 in two enzymes. Moreover, the degradation products by two CbPLs were different. These results revealed these enzymes could represent a potential pectate lyase for applications in paper and textile industries.

scholarly journals Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation

2020 ◽  
Vol 86 (16) ◽  
Author(s):  
Hamed I. Hamouda ◽  
Nasir Ali ◽  
Hang Su ◽  
Jie Feng ◽  
Ming Lu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plant Biomass ◽  
Degradation Products ◽  
Amino Acid Identity ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Acid Identity ◽  
Pectate Lyases ◽  
Caldicellulosiruptor Bescii

ABSTRACT Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass. IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii. The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.

scholarly journals A mannanase, ManA, of the polycentric anaerobic fungusOrpinomycessp. strain PC-2 has carbohydrate binding and docking modules

2005 ◽  
Vol 51 (7) ◽  
pp. 559-568 ◽  
Author(s):  
Eduardo A Ximenes ◽  
Huizhong Chen ◽  
Irina A Kataeva ◽  
Michael A Cotta ◽  
Carlos R Felix ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Anaerobic Fungus ◽  
Ph Profile ◽  
Catalytic Module ◽  
Cellulose Binding

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or β-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 °C, but was rapidly inactivated at 60 °C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.Key words: Orpinomyces, anaerobic fungi, mannanase, cellulose-binding module, cellulosome.

scholarly journals Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

2010 ◽  
Vol 76 (8) ◽  
pp. 2582-2588 ◽  
Author(s):  
Yongchao Li ◽  
Diana C. Irwin ◽  
David B. Wilson
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Thermobifida Fusca ◽  
Carbohydrate Binding ◽  
Synergistic Activity ◽  
The Family ◽  
Cellulose Binding ◽  
Increased Activity ◽  
Catalytic Cleft

ABSTRACT Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.

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