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

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02009-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7048-7059 ◽

Cited By ~ 26

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.

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

Biochemical Journal ◽

10.1042/bj20021527 ◽

2003 ◽

Vol 372 (3) ◽

pp. 905-910 ◽

Cited By ~ 18

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.

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Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽

10.3390/md18010025 ◽

2019 ◽

Vol 18 (1) ◽

pp. 25 ◽

Cited By ~ 4

Author(s):

Zhelun Zhang ◽

Luyao Tang ◽

Mengmeng Bao ◽

Zhigang Liu ◽

Wengong Yu ◽

...

Keyword(s):

Specific Activity ◽

Biological Activities ◽

Functional Characterization ◽

Alginate Lyase ◽

Carbohydrate Binding ◽

Enzyme Degradation ◽

Carbohydrate Binding Modules ◽

Catalytic Module ◽

Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

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Expression and Characterization of a GH16 Family β-Agarase Derived from the Marine Bacterium Microbulbifer sp. BN3 and Its Efficient Hydrolysis of Agar Using Raw Agar-Producing Red Seaweeds Gracilaria sjoestedtii and Gelidium amansii as Substrates

Catalysts ◽

10.3390/catal10080885 ◽

2020 ◽

Vol 10 (8) ◽

pp. 885

Author(s):

Ren Kuan Li ◽

Xi Juan Ying ◽

Zhi Lin Chen ◽

Tzi Bun Ng ◽

Zhi Min Zhou ◽

...

Keyword(s):

Enzyme Activity ◽

High Efficiency ◽

Specific Activity ◽

Low Cost ◽

Intermediate Stage ◽

Industrial Applications ◽

Carbohydrate Binding ◽

Gelidium Amansii ◽

Red Seaweeds ◽

Hydrolysis Of

Agarases catalyze the hydrolysis of agarose to oligosaccharides which display an array of biological and physiological functions with important industrial applications in health-related fields. In this study, the gene encoding agarase (Aga-ms-R) was cloned from Microbulbifer sp. BN3 strain. Sequence alignment indicated that Aga-ms-R belongs to the GH16 family and contains one active domain and two carbohydrate binding module (CBM) domains. The mature Aga-ms-R was expressed successfully by employing the Brevibacillus system. Purified rAga-ms-R was obtained with a specific activity of 100.75 U/mg. rAga-ms-R showed optimal activity at 50 °C and pH 7.0, and the enzyme activity was stable at 50 °C and also over the pH range of 5.0–9.0. After exposure of rAga-ms-R to 70 °C for 30 min, only partial enzyme activity remained. Thin layer chromatographic analysis of the enzymatic hydrolysate of agar obtained using rAga-ms-R disclosed that the hydrolysate comprised, in a long intermediate-stage of the hydrolysis reaction, mainly neoagarotetraose (NA4) and neoagarohexaose (NA6) but ultimately, predominantly neoagarotetraose and trace amounts of neoagarobiose (NA2). Hydrolysates of the raw red seaweeds Gracilaria sjoestedtii and Gelidium amansii, produced by incubation with rAga-ms-R, were mainly composed of neoagarotetraose. The results demonstrate the high efficiency of rAga-ms-R in producing neoagaraoligosaccharide under low-cost conditions.

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Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus

Journal of Bacteriology ◽

10.1128/jb.00609-08 ◽

2008 ◽

Vol 190 (24) ◽

pp. 8220-8222 ◽

Cited By ~ 38

Author(s):

Anat Ezer ◽

Erez Matalon ◽

Sadanari Jindou ◽

Ilya Borovok ◽

Nof Atamna ◽

...

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Cellulose Degradation ◽

Glycoside Hydrolases ◽

Rumen Bacterium ◽

Carbohydrate Binding ◽

Ruminococcus Albus ◽

System A ◽

C Terminus ◽

Carbohydrate Binding Modules

ABSTRACT The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

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Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

10.1101/2021.02.04.429793 ◽

2021 ◽

Author(s):

Krzysztof Mikolajczyk ◽

Anna Bereznicka ◽

Katarzyna Szymczak-Kulus ◽

Katarzyna Haczkiewicz-Lesniak ◽

Bozena Szulc ◽

...

Keyword(s):

Enzyme Activity ◽

Subcellular Localization ◽

Posttranslational Modification ◽

Regulatory Mechanism ◽

Specific Activity ◽

Sweet Spot ◽

Dual Nature ◽

Glycosylation Sites ◽

Opposing Effects

AbstractN-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the role of N-glycans in the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Galβ (Gb3 and the P1 antigen) and Galα1→4GalNAcβ disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and a glycoform without it showed increased enzyme activity. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localize in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the ER. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling biological activity of proteins.

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Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

Applied and Environmental Microbiology ◽

10.1128/aem.02634-20 ◽

2020 ◽

pp. AEM.02634-20

Author(s):

Mohamed A. Attia ◽

Harry Brumer

Keyword(s):

Binding Protein ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Substrate Affinity ◽

Carbohydrate Binding ◽

Carbohydrate Active Enzymes ◽

Plant Polysaccharides ◽

New Family ◽

Carbohydrate Binding Modules ◽

Cellvibrio Japonicus

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00523-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5483-5489 ◽

Cited By ~ 8

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.

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Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5787-5793.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5787-5793 ◽

Cited By ~ 20

Author(s):

Kazutoyo Yoda ◽

Atsushi Toyoda ◽

Yoshihiro Mukoyama ◽

Yutaka Nakamura ◽

Hajime Minato

Keyword(s):

Amino Acid ◽

Sequence Similarity ◽

Glycosyl Hydrolase ◽

Amino Acid Sequences ◽

Substrate Affinity ◽

Carbohydrate Binding ◽

Gene Encoding ◽

Carbohydrate Binding Modules ◽

Hydrolytic Activities ◽

Eubacterium Cellulosolvens

ABSTRACT A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01029-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6610-6620 ◽

Cited By ~ 18

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.

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