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|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-Chitinase|
High purity Tetraacetyl-chitotetraose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Prepared from chitin.
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.Hide Abstract
Biochemical characterization of a bifunctional chitinase/lysozyme from Streptomyces sampsonii suitable for N-acetyl chitobiose production.
Zhang, W., Liu, Y., Ma, J., Yan, Q., Jiang, Z. & Yang, S. (2020). Biotechnology Letters, 1-11.
Chitinases play important role in chitin bioconversion, while few of them have been put into use due to their poor properties. We aimed to identify and characterize chitinases suitable for N-acetyl chitooligosaccharides (COSs) production from chitin materials. A chitinase gene (SsChi28) from Streptomyces sampsonii XY2-7 was cloned and heterologously expressed in E. coli BL21 (DE3) as an active protein. The deduced protein shared high sequence identities and structure similarities with some glycoside hydrolase family 19 chitinases. The recombinant enzyme (SsChi28) was purified and biochemically characterized. SsChi28 was a monomeric protein with a molecular mass of 30 kDa estimated by SDS-PAGE. It was most active at pH 6.0 and 55°C, respectively, and stable in a wide pH range of 3.5-11.5 and up to 60°C. The enzyme exhibited strict substrate specificities towards ethylene glycol chitin (222.3 U/mg) and colloidal chitin (20.1 U/mg). Besides, it displayed lysozyme activity against Micrococcus lysodeikticus. SsChi28 hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose, accounting high up to 73% (w/w) in total products. The excellent enzymatic properties of SsChi28 may make it potential in chitin bioconversion (especially for N-acetyl COS production), as well as in biological control of fungal diseases.Hide Abstract
Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion.
Deng, J. J., Shi, D., Mao, H. H., Li, Z. W., Liang, S., Ke, Y. & Luo, X. C. (2019). International Journal of Biological Macromolecules, 134, 113-121.
In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.Hide Abstract
An actinobacteria lytic polysaccharide monooxygenase acts on both cellulose and xylan to boost biomass saccharification.
Corrêa, T. L. R., Júnior, A. T., Wolf, L. D., Buckeridge, M. S., dos Santos, L. V. & Murakami, M. T. (2019). Biotechnology for Biofuels, 12(1), 117.
Background: Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. Results: The enzyme KpLPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, KpLPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of KpLPMO10A with Celluclast ® and an endo-β-1,4-xylanase also supports this finding. The crystallographic elucidation of KpLPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident diference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. Conclusions: This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic efect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass.Hide Abstract
Comparative biocontrol ability of chitinases from bacteria and recombinant chitinases from the thermophilic fungus Thermomyces lanuginosus.
Okongo, R. N., Puri, A. K., Wang, Z., Singh, S. & Permaul, K. (2019). Journal of Bioscience and Bioengineering, 127(6), 663-671.
Microbial chitinases (EC 220.127.116.11) are known to hydrolyse the chitinous gut epithelium of insects and cell walls of many fungi. In this study, seven chitinases from different bacteria and fungi were produced, characterized and their biocontrol abilities against graminaceous stem borers Eldana saccharina, Chilo partellus and Sesamia calamistis were assessed. All chitinases were stable over broad ranges of pH and temperature, however, recombinant fungal chitinases were more acid-stable than the bacterial counterparts. Chitinases from the thermophilic filamentous fungi Thermomyces lanuginosus SSBP (Chit1) and from Bacillus licheniformis (Chit lic) caused 70% and 80% mortality, respectively, in second instar larvae of E. saccharina. Six of the seven partially-purified microbial chitinases inhibited Aspergillus niger, A. flavus, A. alliaceus, A. ochraceus, Fusarium verticillioides and Mucor sp. Overall, microbial chitinases show promise as biocontrol agents of fungi and stalk–boring lepidopterans.Hide Abstract
Chitooligosaccharide binding to CIA17 (Coccinia indica agglutinin). Thermodynamic characterization and formation of higher order complexes.
Bobbili, K. B., Singh, B., Narahari, A., Bulusu, G., Surolia, A. & Swamy, M. J. (2019). International Journal of Biological Macromolecules, 137, 774-782.
CIA17 is a PP2-like, homodimeric lectin made up of 17 kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)2–6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (Ka = 1.8 × 107 M−1) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)6-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.Hide Abstract
Krolicka, M., Hinz, S. W., Koetsier, M. J., Joosten, R., Eggink, G., van den Broek, L. A. & Boeriu, C. G. (2018). Journal of Agricultural and Food Chemistry, 66(7), 1658-1669.
A thermostable chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40°C (>140 h 90% activity), 50°C (>168 h 90% activity), and 55°C (half-life 48 h). Chitinase Chi1 has broad substrate specificity, and converts chitin, chitosan, modified chitosan and chitin oligosaccharides. The activity of chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw) and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity towards chitin oligosaccharides (GlcNAc)4-6 than towards (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme’s active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.Hide Abstract
Monge, E. C., Tuveng, T. R., Vaaje-Kolstad, G., Eijsink, V. G. & Gardner, J. G. (2018). Journal of Biological Chemistry, jbc-RA117.
Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature, however the complex mechanisms used to detect, degrade, and consume these substrates are not well understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four family 18 Glycoside Hydrolase (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the GH18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.Hide Abstract
An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.
Sabbadin, F., Hemsworth, G. R., Ciano, L., Henrissat, B., Dupree, P., Tryfona, T., Marques, R. D. S., Sweeney, S. T., Besser, K., Elias, L., Pesante, G., Li, Y., Dowle, A. A., Bates, R., Gomez, L. D., Simister, R., Davies, G. J., Walton, P. H., Bruce, N. C. & Pesante, G. (2018). Nature communications, 9(1), 756.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over twenty members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin and was recruited by early arthropods with possible roles in remodelling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes towards cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.Hide Abstract
Krolicka, M., Hinz, S. W., Koetsier, M. J., Eggink, G., van den Broek, L. A. & Boeriu, C. G. (2018). Applied Microbiology and Biotechnology, 102(17), 7441-7454.
Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50°C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55°C (144 h, 75% activity), 60°C (48 h, 85% activity; half-life 82 h), and 70°C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MtHNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.Hide Abstract
Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus.
Mutahir, Z., Mekasha, S., Loose, J. S., Abbas, F., Vaaje‐Kolstad, G., Eijsink, V. G. & Forsberg, Z. (2018). FEBS Letters, 592, 2562-2571.
Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra‐modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin‐type III (FnIII)‐like domains, and a carbohydrate‐binding module (CBM5), is a powerful chitin‐active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin.Hide Abstract
Berton, P., Shamshina, J. L., Ostadjoo, S., King, C. A. & Rogers, R. D. (2018). Carbohydrate Polymers, 199, 228-235.
Chitin, one of Nature’s most abundant biopolymers, can be obtained by either traditional chemical pulping or by extraction using the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate. The IL extraction and coagulation process provides access to a unique chitin, with an open hydrated gel-like structure. Here, enzymatic hydrolysis of this chitin hydrogel, dried shrimp shell, chitin extracted from shrimp shells using IL and then dried, and commercial chitin was carried out using chitinase from Streptomyces griseus. The enzymatic hydrolysis of shrimp shells resulted only in the monomer N-acetylglucosamine, while much higher amounts of the dimer (N, N′-diacetylchitobiose) compared to the monomer were detected when using all forms of ‘pure’ chitin. Interestingly, small amounts of the trimer (N, N′,N′′-triacetylchitotriose) were also detected when the IL-chitin hydrogel was used as substrate. Altogether, our findings indicate that the product distribution and yield are highly dependent on the substrate selected for the reaction and its hydrated state.Hide Abstract
Nareddy, P. K. & Swamy, M. J. (2017). International Journal of Biological Macromolecules, In Press.
In plants, chitooligosaccharide-binding phloem exudate lectins play an important role in the defense mechanism against parasites. Here, we investigated the thermal and chaotrope-induced unfolding of cucumber (Cucumis sativus) phloem exudate lectin (CPL). Circular dichroism (CD) spectroscopic studies indicate that the secondary and tertiary structures of CPL are essentially unaltered up to 90°C. Consistent with this, differential scanning calorimetric studies revealed that CPL is highly thermostable and undergoes a cooperative thermal unfolding transition centered at 97.6°C. The unfolding process was calorimetrically irreversible, and could be described by a non-two-state model, suggesting that upon undergoing a reversible unfolding transition the protein attains a final state in an irreversible step. The ratio of calorimetric and van’t Hoff enthalpies (ΔHc/ΔHv/) was >1.0, suggesting that the two monomers in the dimeric protein unfold at the same temperature. CD spectra recorded at different pH indicated that the secondary and tertiary structures of the protein are nearly unaltered in the pH range 3.0-10.0. Guanidine hydrochloride-induced unfolding studies indicate that chemical denaturation of CPL can also be described by a two-state process, without involving any intermediate. The stability of CPL to high temperatures and large variations of pH appear to be particularly suited for its role in plant defense.Hide Abstract
Moon, C., Seo, D. J., Song, Y. S., Hong, S. H., Choi, S. H. & Jung, W. J. (2017). Microbial Pathogenesis, 113, 218-224.
Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta Americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidalchitin medium at 30°C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography(TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)2, N-acetyl-chitin trimer (GlcNAc)3, and N-acetyl-chitin tetramer (GlcNAc)4 were degraded to (GlcNAc)1-3 on a TLC plate. In an additional experiment, (GlcNAc)6 was degraded to (GlcNAc)1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50°C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred with 50°C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.Hide Abstract
Regel, E. K., Weikert, T., Niehues, A., Moerschbacher, B. M. & Singh, R. (2017). Biotechnology and Bioengineering, In Press.
Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme.Hide Abstract
Tuveng, T. R., Rothweiler, U., Udatha, G., Vaaje-Kolstad, G., Smalås, A. & Eijsink, V. G. (2017). PloS One, 12(11), e0187544.
Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.Hide Abstract
Nareddy, P. K., Bobbili, K. B. & Swamy, M. J. (2017). International Journal of Biological Macromolecules, 95, 910-919.
A chitooligosaccharide-specific lectin has been purified from the phloem exudate of cucumber (Cucumis sativus) by affinity chromatography on chitin. The molecular weight of the cucumber phloem lectin (CPL) was determined as 51912.8 Da by mass spectrometry whereas SDS-PAGE yielded a single band with a subunit mass of 26 kDa, indicating that the protein is a homodimer. Peptide mass fingerprinting studies strongly suggest that CPL is identical to the 26 kDa phloem protein 2 (PP2) from cucumber. CD spectroscopy indicated that CPL is a predominantly β-sheets protein. Hemagglutination activity of CPL was mostly unaffected between 4 and 90°C and between pH 4.0 and 10.0, indicating functional stability of the protein. Isothermal titration calorimetric studies indicate that the CPL dimer binds to two chitooligosaccharide ((GlcNAc)2-6) molecules with association constants ranging from 1.0 × 103 to 17.5 × 105 M-1. The binding reaction was strongly enthalpy driven (δHb = −ve) with negative contribution from binding entropy (δSb = −ve). The enthalpy-driven nature of binding reactions suggests that hydrogen bonding and van der Waals interactions stabilize the CPL-chitooligosaccharide association. Enthalpy-entropy compensation was observed for the CPL-chitooligosaccharide interaction, indicating that water molecules play an important role in the binding process.Hide Abstract
Kobayashi, T., Uchimura, K., Kubota, T., Nunoura, T. & Deguchi, S. (2016). Applied Microbiology and Biotechnology, 100(1), 203-214.
A β-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80°C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845–7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.Hide Abstract
Tobaruela, E. D. C., Santos, A. D. O., de Almeida-Muradian, L. B., Araujo, E. D. S., Lajolo, F. M. & Menezes, E. W. (2016). Food Chemistry.
AOAC 2011.25 method enables the quantification of most of the dietary fiber (DF) components according to the definition proposed by Codex Alimentarius. This study aimed to compare the DF content in fruits analyzed by the AOAC 2011.25 and AOAC 991.43 methods. Plums (Prunus salicina), atemoyas (Annona x atemoya), jackfruits (Artocarpus heterophyllus), and mature coconuts (Cocos nucifera) from different Brazilian regions (3 lots/fruit) were analyzed for DF, resistant starch, and fructans contents. The AOAC 2011.25 method was evaluated for precision, accuracy, and linearity in different food matrices and carbohydrate standards. The DF contents of plums, atemoyas, and jackfruits obtained by AOAC 2011.25 was higher than those obtained by AOAC 991.43 due to the presence of fructans. The DF content of mature coconuts obtained by the same methods did not present a significant difference. The AOAC 2011.25 method is recommended for fruits with considerable fructans content because it achieves more accurate values.Hide Abstract
Hamre, A. G., Schaupp, D., Eijsink, V. G. & Sørlie, M. (2015). FEBS Letters, 589(15), 1807-1812.
The enzymatic degradation of the closely related insoluble polysaccharides; cellulose (β(1–4)-linked glucose) by cellulases and chitin (β(1–4)-linked N-acetylglucosamine) by chitinases, is of large biological and economical importance. Processive enzymes with different inherent directionalities, i.e. attacking the polysaccharide chains from opposite ends, are crucial for the efficiency of this degradation process. While processive cellulases with complementary functions differ in structure and catalytic mechanism, processive chitinases belong to one single protein family with similar active site architectures. Using the unique model system of Serratia marcescens with two processive chitinases attacking opposite ends of the substrate, we here show that different directionalities of processivity are correlated to distinct differences in the kinetic signatures for hydrolysis of oligomeric tetra-N-acetyl chitotetraose.Hide Abstract
Hamre, A. G., Eide, K. B., Wold, H. H. & Sørlie, M. (2015). Carbohydrate Research, 407, 166-169.
For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent kcat (kcatapp) values on a β-chitin substrate. kcatapp values were determined to be
1.7±0.1 s-1 and 1.7±0.1 s-1 for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s-1 for the endo-active ChiC. The addition of CBP21 boosted the kcatapp values of ChiA and ChiB giving values of 11.1±1.5 s-1 and 13.9±1.4 s-1, while there was no effect on ChiC (0.9±0.1 s-1).
Takasuka, T. E., Bianchetti, C. M., Tobimatsu, Y., Bergeman, L. F., Ralph, J. & Fox, B. G. (2014). Proteins: Structure, Function, and Bioinformatics, 82(7), 1245-1257.
SACTE_5457 is secreted by Streptomyces sp. SirexAA-E, a highly cellulolytic actinobacterium isolated from a symbiotic community composed of insects, fungi, and bacteria. Here we report the 1.84 Å resolution crystal structure and functional characterization of SACTE_5457. This enzyme is a member of the glycosyl hydrolase family 46 and is composed of two α-helical domains that are connected by an α-helical linker. The catalytic residues (Glu74 and Asp92) are separated by 10.3 Å, matching the distance predicted for an inverting hydrolysis reaction. Normal mode analysis suggests that the connecting α-helix is flexible and allows the domain motion needed to place active site residues into an appropriate configuration for catalysis. SACTE_5457 does not react with chitin, but hydrolyzes chitosan substrates with an ~4-fold improvement in kcat/KM as the percentage of acetylation and the molecular weights decrease. Analysis of the time dependence of product formation shows that oligosaccharides with degree of polymerization N-acetyl-D-glucosamine in the +2 subsite and may weakly interfere with binding of N-acetyl-D-glucosamine in the +1 subsites. A proposal for how these constraints account for the observed product distributions is provided.Hide Abstract
Nguyen, H. A., Nguyen, T. H., Křen, V., Eijsink, V. G. H., Haltrich, D. & Peterbauer, C. K. (2012). Journal of Agricultural and Food Chemistry, 60(12), 3275-3281.
The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. Km and Kcat for pNP-GlcNAc were 2.56 mM and 26.7 s-1, respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the Km being very high (>10 mM). The optimal temperature and pH of the enzyme were 37°C and 5.5, respectively, for both substrates. The half-life of activity at 37°C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50°C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5–8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA’s role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.Hide Abstract
Chang, Y. L., Liao, S. K. S., Chen, Y. C., Hung, W. T., Yu, H. M., Yang, W. B., Fang, J. M., Chen, C. H. & Lee, Y. C. (2011). Journal of mass spectrometry, 46(3), 247-255.
MALDI-MS provides a rapid and sensitive analysis of large biomolecules such as proteins and nucleic acids. However, oligo- and polysaccharides are less sensitive in MS analysis partly due to their neutral and hydrophilic nature to cause low ionization efficiency. In this study, four types of oligosaccharides including aldoses, aminoaldoses, alduronic acids and α-keto acids were modified by appropriate tags at the reducing termini to improve their ionization efficiency. Bradykinin (BK), a vasoactive nonapeptide (RPPGFSPFR), containing two arginine and two phenylalanine residues turned out to be an excellent MS signal enhancer for maltoheptaose, GlcNAc oligomers and oligogalacturonic acids. In the MALDI-TOF-MS analysis using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix, the GalA4–BK and GalA5–BK conjugates prepared by reductive amination showed the detection limit at 0.1 fmol, i.e. ∼800-fold enhancement over the unmodified pentagalacturonic acids. The remarkable MS enhancement was attributable to the synergistic effect of the basic arginine residues for high proton affinity and the hydrophobic property phenylalanine residues for facile ionization. A tetrapeptide GFGR(OMe) and an arginine linked phenylenediamine (H2N)2Ph-R(OMe) were thus designed to act as potent tags of oligosaccharides in MS analysis. Interestingly, concurrent condensation and lactonization of α2,8-linked tetrasialic acid (SA4) was carried out with (H2N)2Ph-R(OMe) to obtain a quinoxalinone derivative, which showed > 200-fold enhancement over unmodified SA4 in the MALDI-TOF-MS analysis.Hide Abstract