scholarly journals Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22

3 Biotech ◽  
2021 ◽  
Vol 11 (12) ◽  
Author(s):  
Goutam Mohan Pawaskar ◽  
Keyur Raval ◽  
Prathibha Rohit ◽  
Revathi P. Shenoy ◽  
Ritu Raval
Keyword(s):  
Biomedical Application ◽  
Marine Ecosystem ◽  
Hydrolytic Enzyme ◽  
Enzyme Characterization ◽  
Relative Activity ◽  
Chitin Deacetylase ◽  
Factor Screening ◽  
Bacillus Aryabhattai ◽  
Chitosan Oligosaccharides ◽  
Thermo Stability

AbstractChitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E−05 µg mL−1, 3.06E + 01 µM mg−1 min−1 and 3.27E + 04 s−1, respectively, with EGC as the substrate and 7.14E−07 µg mL−1, 7.14E + 01 µM mg−1 min−1 and 1.40E + 06 s−1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application.

scholarly journals Quorum Sensing Regulates the Hydrolytic Enzyme Production and Community Composition of Heterotrophic Bacteria in Coastal Waters

2021 ◽  
Vol 12 ◽  
Author(s):  
Marion Urvoy ◽  
Raphaël Lami ◽  
Catherine Dreanno ◽  
Daniel Delmas ◽  
Stéphane L’Helguen ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Community Composition ◽  
Coastal Waters ◽  
Bacterial Communities ◽  
Heterotrophic Bacteria ◽  
Bacterial Community Composition ◽  
Marine Ecosystem ◽  
Hydrolytic Enzyme ◽  
Enzymatic Activities ◽  
Hydrolytic Activities

Heterotrophic microbial communities play a central role in biogeochemical cycles in the ocean by degrading organic matter through the synthesis of extracellular hydrolytic enzymes. Their hydrolysis rates result from the community’s genomic potential and the differential expression of this genomic potential. Cell-cell communication pathways such as quorum sensing (QS) could impact both aspects and, consequently, structure marine ecosystem functioning. However, the role of QS communications in complex natural assemblages remains largely unknown. In this study, we investigated whether N-acylhomoserine lactones (AHLs), a type of QS signal, could regulate both hydrolytic activities and the bacterial community composition (BCC) of marine planktonic assemblages. To this extent, we carried out two microcosm experiments, adding five different AHLs to bacterial communities sampled in coastal waters (during early and peak bloom) and monitoring their impact on enzymatic activities and diversity over 48 h. Several specific enzymatic activities were impacted during both experiments, as early as 6 h after the AHL amendments. The BCC was also significantly impacted by the treatments after 48 h, and correlated with the expression of the hydrolytic activities, suggesting that changes in hydrolytic intensities may drive changes in BCC. Overall, our results suggest that QS communication could participate in structuring both the function and diversity of marine bacterial communities.

scholarly journals Imobilisasi Enzim Lipase Dedak Padi (Oryza Sativa L.) Pada Karbon Aktif: Karakterisasi, dan Uji Stabilitas Kerja Enzim Imobil

2017 ◽  
Vol 5 (1) ◽  
pp. 32-36 ◽  
Author(s):  
Firdaus Firdaus ◽  
Seniwati Dali ◽  
Hendra J. Rusman
Keyword(s):  
Thermal Stability ◽  
Oryza Sativa L ◽  
Carbon Matrix ◽  
Enzyme Characterization ◽  
Relative Activity ◽  
Ph Optimum ◽  
Repeated Use ◽  
The Stability ◽  
Matrix Enzyme

This research aims to immobilization; characterize the enzyme of immobilized, test the effectiveness of the enzyme of immobilized. This research begins with the immobilization to process of enzyme lipase using activated carbon matrix, enzyme characterization covering of immobile determination of temperature and pH optimum of the enzyme of immobilized, as well as test the stability of work covering immobilized of enzyme the test thermal stability and repeated use. The results showed that the immobile of enzyme work optimally at 50oC of temperature and pH 6.5 with each activity 0.040 U/mL; research results also showed that the immobile of enzyme has higher thermal stability in comparison with the free enzyme: with the relative activity of 57.50% at the time of 45 minutes of exposure and the exposure time at 47.50% at 75-105 minutes and it can be used as many as six times with the relative activity of 52.5% in 6 times of use.

scholarly journals Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides

Marine Drugs ◽  
2019 ◽  
Vol 17 (2) ◽  
pp. 74 ◽  
Author(s):  
Xian-Yu Zhu ◽  
Yong Zhao ◽  
Huai-Dong Zhang ◽  
Wen-Xia Wang ◽  
Hai-Hua Cong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mode Of Action ◽  
High Performance ◽  
Biological Activities ◽  
Ionization Mass ◽  
Chitin Deacetylase ◽  
Specific Mode ◽  
Chitosan Oligosaccharides ◽  
Function Relationship ◽  
And Function

Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from Saccharomyces cerevisiae, ScCDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. ScCDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that ScCDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. ScCDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore ScCDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

scholarly journals A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

2016 ◽  
Vol 82 (22) ◽  
pp. 6645-6655 ◽  
Author(s):  
Shoa Naqvi ◽  
Stefan Cord-Landwehr ◽  
Ratna Singh ◽  
Frank Bernard ◽  
Stephan Kolkenbrock ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Biologically Active ◽  
Puccinia Graminis ◽  
Chitin Deacetylase ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
Potential Applications ◽  
Random Patterns

ABSTRACTPartially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemicalN-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungusPuccinia graminisf. sp.triticiinEscherichia coli. The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein fromP. graminis(PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed thatPgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating thatPgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property ofPgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.IMPORTANCEWe successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycetePuccinia graminisf. sp.triticiin the periplasm ofE. colias a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

scholarly journals Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases

2020 ◽  
Vol 19 (12) ◽  
pp. 2104-2114
Author(s):  
Matthias Schittmayer ◽  
Nemanja Vujic ◽  
Barbara Darnhofer ◽  
Melanie Korbelius ◽  
Sophie Honeder ◽  
...  
Keyword(s):  
Small Intestine ◽  
Lipid Droplets ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Relative Activity ◽  
Proximal Jejunum ◽  
Serine Hydrolase ◽  
Spatially Resolved ◽  
Molecular Events ◽  
Labeling Approach

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization

2018 ◽  
Vol 90 (8) ◽  
pp. 5209-5216 ◽  
Author(s):  
Nathaniel E. Kallmyer ◽  
Joseph Musielewicz ◽  
Joel Sutter ◽  
Nigel F. Reuel
Keyword(s):  
Carbon Nanotube ◽  
Hydrolytic Enzyme ◽  
Enzyme Characterization ◽  
Single Walled Carbon Nanotube ◽  
Single Walled Carbon

Spatial and temporal dynamics of Pacific capelin Mallotus catervarius in the Gulf of Alaska: implications for ecosystem-based fisheries management

2020 ◽  
Vol 637 ◽  
pp. 117-140 ◽  
Author(s):  
DW McGowan ◽  
ED Goldstein ◽  
ML Arimitsu ◽  
AL Deary ◽  
O Ormseth ◽  
...  
Keyword(s):  
Temporal Dynamics ◽  
Marine Ecosystem ◽  
Gulf Of Alaska ◽  
Data Series ◽  
Limited Information ◽  
Important Species ◽  
Multiple Data ◽  
Spatial And Temporal Dynamics ◽  
Spawning Areas ◽  
Spatio Temporal

Pacific capelin Mallotus catervarius are planktivorous small pelagic fish that serve an intermediate trophic role in marine food webs. Due to the lack of a directed fishery or monitoring of capelin in the Northeast Pacific, limited information is available on their distribution and abundance, and how spatio-temporal fluctuations in capelin density affect their availability as prey. To provide information on life history, spatial patterns, and population dynamics of capelin in the Gulf of Alaska (GOA), we modeled distributions of spawning habitat and larval dispersal, and synthesized spatially indexed data from multiple independent sources from 1996 to 2016. Potential capelin spawning areas were broadly distributed across the GOA. Models of larval drift show the GOA’s advective circulation patterns disperse capelin larvae over the continental shelf and upper slope, indicating potential connections between spawning areas and observed offshore distributions that are influenced by the location and timing of spawning. Spatial overlap in composite distributions of larval and age-1+ fish was used to identify core areas where capelin consistently occur and concentrate. Capelin primarily occupy shelf waters near the Kodiak Archipelago, and are patchily distributed across the GOA shelf and inshore waters. Interannual variations in abundance along with spatio-temporal differences in density indicate that the availability of capelin to predators and monitoring surveys is highly variable in the GOA. We demonstrate that the limitations of individual data series can be compensated for by integrating multiple data sources to monitor fluctuations in distributions and abundance trends of an ecologically important species across a large marine ecosystem.

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