The Function of CBM32 in Alginate Lyase VxAly7B on the Activity on Both Soluble Sodium Alginate and Alginate Gel

Carbohydrate-binding modules (CBMs), as an important auxiliary module, play a key role in degrading soluble alginate by alginate lyase, but the function on alginate gel has not been elucidated. Recently, we reported alginate lyase VxAly7B containing a CBM32 and a polysaccharide lyase family 7 (PL7). To investigate the specific function of CBM32, we characterized the full-length alginate lyase VxAly7B (VxAly7B-FL) and truncated mutants VxAly7B-CM (PL7) and VxAly7B-CBM (CBM32). Both VxAly7B-FL and native VxAly7B can spontaneously cleavage between CBM32 and PL7. The substrate-binding capacity and activity of VxAly7B-CM to soluble alginate were 0.86- and 1.97-fold those of VxAly7B-FL, respectively. Moreover, CBM32 could accelerate the expansion and cleavage of alginate gel beads, and the degradation rate of VxAly7B-FL to alginate gel beads was threefold that of VxAly7B-CM. Results showed that CBM32 is not conducive to the degradation of soluble alginate by VxAly7B but is helpful for binding and degradation of insoluble alginate gel. This study provides new insights into the function of CBM32 on alginate gel, which may inspire the application strategy of CBMs in insoluble substrates.

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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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The fixation of a water-soluble drug in calcium-induced alginate gel beads and the release profiles from the vehicle.

Drug Delivery System ◽

10.2745/dds.12.49 ◽

1997 ◽

Vol 12 (1) ◽

pp. 49-55 ◽

Cited By ~ 2

Author(s):

Yoshifumi Murata ◽

Norie Katayana ◽

Takashi Kajita ◽

Etsuko Miyamoto ◽

Susumu Kawashima

Keyword(s):

Water Soluble ◽

Alginate Gel ◽

Gel Beads ◽

Water Soluble Drug ◽

Release Profiles

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Nitrification and Autotrophic Denitrification in Calcium Alginate Beads

Water Science & Technology ◽

10.2166/wst.1987.0199 ◽

1987 ◽

Vol 19 (1-2) ◽

pp. 175-182 ◽

Cited By ~ 16

Author(s):

Z. Lewandowski ◽

R. Bakke ◽

W. G. Characklis

Keyword(s):

Calcium Carbonate ◽

Calcium Ions ◽

Continuous Flow ◽

Calcium Alginate ◽

Microbial Respiration ◽

Flow System ◽

Alginate Beads ◽

Alginate Gel ◽

Gel Beads ◽

Calcium Alginate Gel

Immobilization of nitrifiers and autotrophic denitrifiers (Thiobacillus denitrificans) within calcium alginate gel was demonstrated. Calcium carbonate reagent was immobilized along with bacteria as the stabilizing agent. Protons released as a result of microbial respiration reacted with calcium carbonate producing calcium ions which internally stabilized the calcium alginate gel. The microbially active gel beads were mechanically stable and active for three months in a continuous flow system without addition of calcium.

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Structural basis for the exolytic activity of polysaccharide lyase family 6 alginate lyase BcAlyPL6 from human gut microbe Bacteroides clarus

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2021.02.040 ◽

2021 ◽

Vol 547 ◽

pp. 111-117

Author(s):

Bing Wang ◽

Sheng Dong ◽

Fu-Li Li ◽

Xiao-Qing Ma

Keyword(s):

Alginate Lyase ◽

Structural Basis ◽

Human Gut ◽

Polysaccharide Lyase ◽

Gut Microbe

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Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100638 ◽

2021 ◽

pp. 100638

Author(s):

Marie Sofie Møller ◽

Souad El Bouaballati ◽

Bernard Henrissat ◽

Birte Svensson

Keyword(s):

Functional Diversity ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules

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Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials ◽

10.3390/ma14123175 ◽

2021 ◽

Vol 14 (12) ◽

pp. 3175

Author(s):

Mariana Barbosa ◽

Hélvio Simões ◽

Duarte Miguel F. Prazeres

Keyword(s):

Fusion Proteins ◽

Fluorescent Protein ◽

Protein A ◽

Chemical Properties ◽

Main Idea ◽

Bioactive Molecules ◽

Scaffolding Protein ◽

Carbohydrate Binding ◽

Biological Interactions ◽

Carbohydrate Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes

Scientific Reports ◽

10.1038/s41598-019-53173-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hong-Hsing Liu ◽

Yu-Chen Lin ◽

Chen-Shuan Chung ◽

Kevin Liu ◽

Ya-Hui Chang ◽

...

Keyword(s):

Markov Models ◽

Protein Domains ◽

Degradation Pathway ◽

The Other ◽

Carbohydrate Binding ◽

Gram Stain ◽

Active Protein ◽

Metabolic Potential ◽

Independent Validation ◽

Carbohydrate Binding Modules

AbstractBowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10−4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10−5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

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