Wikipedia

N-acetyl-β-d-glucosaminidase

N-acetyl-β-d-glucosaminidase (EC 3.2.1.30; EC 3.2.1.52) is a mesophilic hydrolase that specifically hydrolyzes N-acetyl-glucosides. The enzyme is found across a wide variety of marine and terrestrial creatures with the primary function of breaking down oligosaccharides in the presence of water. One of the primary functions of the enzyme is to target and hydrolyze oligosaccharides containing chitin. In this chitinase function, the enzyme contributes to the ability of many organisms to break down chitin-containing molecules and subsequently digest or re-uptake environmental chitin, carbon, or nitrogen. The enzyme's crystal structure varies slightly across organisms, but is characterized by three or four domains with one active site. Across proteins, the active site entails an α-β barrel with either an arginine or tryptophan residues in the barrel pocket to bind incoming substrate.

Enzyme Commission (EC) nomenclature

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N-acetyl-β-d-glucosaminidase is often referred to under one of two EC numbers, depending on the synonym used in the literature. EC 3.2.1.30 refers to the single n-acetyl-β-d-glucosaminidase enzyme, which is one of four in a larger enzyme complex.[1][2][3][4] EC 3.2.1.52 refer to β-n-acetyl-hexosaminidase, a complex of 4 enzymes including n-acetyl-β-d-glucosaminidase.[1][2][3][4]

EC number meaning

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The various numbers in the EC sequence describe the enzyme function in ascending order of specificity:[2]

  • EC 3. refers to a hydrolase: an enzyme that catalyzes hydrolysis, or a decomposition reaction with water as one of the reactants.[5]
  • EC 3.2 refers to a glycosylase: an enzyme that specifically hydrolyses glycosyl compounds.
  • EC 3.2.1 refers to a glycosidase: an enzyme that hydrolyses oxygen- and sulfur-glycosol compounds.
  • EC 3.2.1.30 refers to n-acetyl-β-d-glucosaminidase: an enzyme that hydrolyses N-acetyl-glucosides.
  • EC 3.2.1.52 refers to β-n-acetyl-hexosaminidase, a 4-enzyme family that has the ability to hydrolyze terminal non-reducing n-acetyl-hexosamine residues (n-acetyl-glucosides and n-acetyl-galactosides).

Synonyms

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In addition to its EC numbers, n-acetyl-β-d-glucosaminidase can be referred by several synonyms in the literature. These include:[6]

  • β-n-acetylglucosaminidase
  • β-acetylaminodeoxyglucosidase
  • β-acetamidodeoxyglucosidase
  • β-acetylglucosaminidase
  • n-acetyl-β-glucosaminidase
  • n-acetyl-β-d-glucosaminidase
  • chitobiase
  • acetyl-β-glucosaminidase
  • β-d-glucosaminidase
  • β-n-acetyl-d-glucosaminidase
  • β-n-acetylaminodeoxyglucosidase
  • exo-n-acetyl-β-d-glucosaminidase
  • p-nitrophenyl-β-n-acetylglucosaminidase
  • exochitinase
  • β-d-n-acetylglucosaminidase

Reaction pathways

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The general reaction structure occurs where, in the presence of water, n-acetyl-β-d-glucosaminidase breaks oligosaccharides into smaller sugar units.[7] More specifically, n-acetyl-β-d-glucosaminidase hydrolyses terminal non-reducing n-acetyl-β-glucosamine residues in chitin molecules (ie chitobiose, chitotriose) and in glycoproteins.[6] Bonds broken during hydrolysis include the β-glycosidic bonds of β-glucosaminide and β-galactosiminide, and specific monosaccharide products include N-acetyl-d-glucosamine and N-acetyl-d-galactosamine.[7][8] N-acetyl-β-d-glucosaminidase has also been observed to catalyze transglycosylation, which in turn facilitates the creation of new oligosaccharides with different amino residues.[7][9] Furthermore, n-acetyl-β-d-glucosaminidase is believed to rely on the given substrate to provide the nucleophile needed to instigate hydrolysis.[10]

Common substrates

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Common substrates utilized in reactions with n-acetyl-β-d-glucosaminidase include:[6]

  • p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside and H2O
  • p-nitrophenyl-2-acetamido-2-deoxy-β-d-galactopyranoside and H2O
  • n, n'-diacetylchitobiose and H2O
  • 4-methylumbelliferyl-β-d-glucosaminide (or galactoaminide) and H2O

Common products

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Common products formed in reactions with n-acetyl-β-d-glucosaminidase include:[6]

  • n-acetylglucosamine and p-nitrophenol
  • n-acetylgalactosamine and p-nitrophenol
  • n-acetylglucosamine
  • 4-methylumbelliferone and n-acetylglucosamine

Km

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The Km of n-acetyl-β-d-glucosaminidase has been reported at values ranging from 0.096 mM in marine fungi to 0.27 mM in Aeromonas sp. for the hydrolysis of p-nitrophenyl-β-d-n-acetylglucosaminide.[7][11] The same reaction catalyzed by proteins isolated from calf brain was generated at an observed Km of 0.72 mM, with a maximum reaction velocity of 2.5 μmoles/mg per hour.[8]

Inhibitors

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Product saturation

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One means by which n-acetyl-β-d-glucosaminidase reactions are inhibited is by increased saturation of reaction product. Hydrolysis of oligosaccharides (ie, chitobiose and chitotriose) to mono- and disaccharides decreases in rate as substrate polymerization level increase (i.e., for chitooligosaccharides with degrees of polymerization between 5 and 8).[7] Similarly, increased concentrations of monosaccharides (N-acetyl-D-glucosamine, glucose, galactose) in the reaction solution can decrease activity by 12-70%.[7][12]

Ionic inhibitors

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Multiple ions have been identified as inhibiting n-acetyl-β-d-glucosaminidase activity. These include:[6][7]

  • Ag+
  • Cu2+
  • Hg2+
  • Zn2+
  • Fe3+
  • Ca2+
  • Cd2+

Other inhibitors

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Other molecular compounds have been observed to depress n-acetyl-β-d-glucosaminidase activity. These include:[6][7]

  • CaCl2
  • MgSO4
  • 2-deoxy-2-acetamido-d-glucono-1
  • 5-lactone
  • iodeacetamide
  • p-chloromercuribenzoate
  • p-aminophenyl-1-thio-β-L-fucopyranoside
  • n-acetylmuramic acid
  • acetate

Organisms that produce and use n-acetyl-β-d-glucosaminidase

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N-acetyl-β-d-glucosaminidase has been recorded and observed in various terrestrial and marine organisms, ranging from bacteria to megafauna. Its activity has been documented extensively in mammals,[4][8] fungi,[7][13][14] crustaceans,[15][16] cartilaginous fishes,[17][18][19] mollusks,[20] jellyfish,[21] and bacteria.[3][10][12][22][23] The broader function of the enzyme within the organism is the breakdown and re-uptake of chitin molecules obtained from either external consumption in the environment or internal growth within the organism. The enzyme has also been observed to play a key role in contributing to local nitrogen levels exuded by microbial communities.[14] Moreover, this enzyme is defined as a marker for the detection of acute kidney injury - it is present in the lysosomes of proximal tubular cells and is not filtered by the glomerulus, but is rapidly cleared from the bloodstream by the liver.[24] Specific examples of how n-acetyl-β-d-glucosaminidase functions within various organisms are provided in the sections below.

Digestion

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In marine species that consume crustacean and/or plant materials that contain chitin, such as bonnethead sharks (Sphyrna tiburo) or Antarctic krill (Euphausia superba), n-acetyl-β-d-glucosaminidase helps breakdown chitin molecules for digestion. For Antarctic krill, the enzyme is generated within the cytosol of the organism's cells and subsequently released into the gut during digestion.[15] In bonnethead sharks, on the other hand, the enzymes are not actually produced by the shark itself but instead by colonies of bacteria within the anterior regions of the animal's intestine. The presence of these enzymes and their function as chitinases allow bonnetheads to consume both crustaceans as well as some plant material, making them one of the few if not only omnivorous species of shark.[17][18] Some species of marine bacteria, such as Vibrio furnissii, utilize n-acetyl-β-d-glucosaminidase to hydrolyze p-nitrophenol (PNP)-β-GlcNAc and 4-methylumbelliferone,7-hydroxy-4-methylcoumarin (MUF)-β-GlcNAc for further digestion. Here the enzyme plays a critical step allowing the bacteria to access nitrogen and carbon from the local marine environment.[22]

Shell maintenance and molting

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For marine organisms that have chitinous shells, such as krill or shrimp species, n-acetyl-β-d-glucosaminidase plays a prominent role in facilitating molting. In the Antarctic krill, northern krill (Meganyctiphanes norvegica), and common prawn (Palaemon serratus), the enzyme breaks terminal N-acetyl-glucosamine monomers from chitin molecules released during initial shell breakdown at the start of the molting period. During the duration of the molting periods, these monomers are continually transported by vesicles closer to the epidermis, below the newly forming shell cuticle, for reabsorption via pore channels and reutilization in the new shell post-molt.[15][16]

Nitrogen remineralization in soils

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When released by fungi and soil microbes, n-acetyl-β-d-glucosaminidase plays a pivotal role increasing soil nitrogen remineralization rates by breaking down chitin into amino sugars for remineralization. The activity of n-acetyl-β-d-glucosaminidase (measured in mg of p-nitrophenol per kilogram soil per hour) has been found to significantly correlate to increased total nitrogen mineralization, organic carbon production, and ammonium fixation in local environments. Because of the high levels of chitin in soil compounds globally, this ability of n-acetyl-β-d-glucosaminidase suggests that the enzyme is a key player in transitioning concentrations of nitrogen, carbon, and ammonium in soil and soil-dependent ecosystems of plants, animals, and microorganisms.[14][23]

Protein structure

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Thermal and pH stability

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N-acetyl-β-d-glucosaminidase is a mesophile, with observed structural and functional stability between 40 and 55 degrees Celsius.[7][20] Maximum activity was recorded at 45 degrees Celsius.[7][20] The enzyme displayed stable structure and function between 3.8 and 6.0 pH, with maximum activity at 4.5 pH and an isoelectric point (a point of no charge on the protein surface) at 4.83 pH.[7][20]

Molecular weight

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Measurements of the molecular weight of n-acetyl-β-d-glucosaminidase range from 68,000 g/mol (observed in proteins isolated from marine fungus, Penicillium canescens) to ~100,000 g/mol (observed in proteins isolated from Aeromonas sp.).[7][11]

Crystal structure

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While the structural features of n-acetyl-β-d-glucosaminidase are not well described for specific proteins isolated across the diversity of organisms it has been identified to function within, the crystal structure and active sites are well-mapped in enzymes obtained from several species of bacteria.

Streptococcus gordonii

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N-acetyl-β-d-glucosaminidase isolated from Streptococcus gordonii was identified as containing 626 amino acids with a three-domain dimer structure. Each monomer within the dimer contains seven cysteine residues and no disulphide bonds. Much of the dimer surface contains hydrophobic residues. Two crystal forms have been observed, one defined by symmetric monomers and the other by asymmetric monomers.[10] The first domain (N-terminal) contains amino residues 2-82. This domain is defined by an α-β fold containing five strands of parallel β-pleated sheet that envelop two α-helices.[10] The second domain contains amino residues 83-400. This domain contains the active site within a triose phosphate isomerase (TIM) barrel at the C-terminal end of the domain. The mouth of the barrel is ringed by eight α-helices, and thee six loops of the barrel structure interact with the second monomer. Barrel conformation differs slightly between the symmetric and asymmetric monomer crystal forms.[10] Lastly, the third domain contains amino residues 401-627. This domain is dominated by α-helices, with five helices adjacent to the active site barrel and in antiparallel configuration to each other.[10]

The dual crystal forms of n-acetyl-β-d-glucosaminidase in Streptococcus gordonii are thought to facilitate the enzymes function through the configuration of active vs. dormant forms. The first crystal form is characterized by the presence of catalytic acids on the ends of the active site barrel's β-pleats, with these acids directly extending towards the inner barrel's tryptophan residues. This conformation is thought to be more favorable for directing incoming oligosaccharides into the active site for hydrolysis. In contrast, the second crystal form has multiple β-pleats turning away from the barrel and opening the active site to solvent, potentially disabling efficient hydrolysis from taking place.[10]

Serratia marcescens

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The crystal structure of n-acetyl-β-d-glucosaminidase isolated from Serratia marcescens bacteria were observed containing a single active site within the enzyme's third domain of four, characterized by an α-β barrel fold.[3][25]

Escheria coli

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N-acetyl-β-d-glucosaminidase cloned and sequenced from Escheria coli were linked to a gene composed of 2655 nucleotides that coded 855 amino acids to yield the final protein. For this enzyme, four domains were identified, where again, the third domain contained the active site within an eight-stranded α-β barrel fold. The first and fourth domains contain two β-pleated sheets, while the second domain is composed of both α-helices and β-pleated sheets.[26] Similar to the crystal structure described for Streptococcus gordonii, the structures present in the enzymes for Escheria coli indicate that the substrate fits into the barrel pocket and is bound at its non-reducing terminal sugar. Unlike the active site for Streptococcus gordonii, however, the binding residue in the active site for Escheria coli is arginine rather than tryptophan. Again, the substrate is understood to provide the nucleophile needed to instigate enzyme affinity to the active site.[26]

References

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  1. ^ a b "ExPASy - ENZYME". enzyme.expasy.org. Retrieved 2021-10-21.
  2. ^ a b c "EC 3.2.1.52". iubmb.qmul.ac.uk. Retrieved 2021-10-21.
  3. ^ a b c d Patil, Reetarani S.; Ghormade, Vandana; Deshpande, Mukund V. (April 2000). "Chitinolytic enzymes: an exploration". Enzyme and Microbial Technology. 26 (7): 473–483. doi:10.1016/s0141-0229(00)00134-4. ISSN 0141-0229. PMID 10771049.
  4. ^ a b c Cabezas, J A (1989-08-01). "Some comments on the type references of the official nomenclature (IUB) for β-N-acetylglucosaminidase, β-N-acetylhexosaminidase and β-N-acetylgalactosaminidase". Biochemical Journal. 261 (3): 1059–1060. doi:10.1042/bj2611059b. ISSN 0264-6021. PMC 1138940. PMID 2529847.
  5. ^ "Hydrolase | class of enzymes". Encyclopedia Britannica. Retrieved 2021-10-21.
  6. ^ a b c d e f Schomburg, Dietmar; Salzmann, Margit (1991), Schomburg, Dietmar; Salzmann, Margit (eds.), "N-Acetyl-beta-glucosaminidase", Enzyme Handbook 4, Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 195–201, doi:10.1007/978-3-642-84437-9_22, ISBN 978-3-642-48986-0, retrieved 2021-10-21
  7. ^ a b c d e f g h i j k l m Burtseva, Yu. V.; Sova, V. V.; Pivkin, M. V.; Anastyuk, S. D.; Gorbach, V. I.; Zvyagintseva, T. N. (November 2010). "Distribution of O-glycosylhydrolases in marine fungi of the Sea of Japan and the Sea of Okhotsk: Characterization of exocellular N-acetyl-β-D-glucosaminidase of the marine fungus Penicillium canescens". Applied Biochemistry and Microbiology. 46 (6): 648–656. doi:10.1134/s0003683810060141. ISSN 0003-6838. S2CID 22078840.
  8. ^ a b c Frohwein, Yaacov Zvi; Gatt, Shimon (1967-09-01). "Isolation of β-N-Acetylhexosaminidase, β-N-Acetylglucosaminidase, and β-N-Acetylgalactosaminidase from Calf Brain*". Biochemistry. 6 (9): 2775–2782. doi:10.1021/bi00861a018. ISSN 0006-2960. PMID 6055190.
  9. ^ Weignerová, Lenka; Vavrušková, Petra; Pišvejcová, Andrea; Thiem, Joachim; Křen, Vladimı́r (April 2003). "Fungal β-N-acetylhexosaminidases with high β-N-acetylgalactosaminidase activity and their use for synthesis of β-GalNAc-containing oligosaccharides". Carbohydrate Research. 338 (9): 1003–1008. doi:10.1016/s0008-6215(03)00044-2. ISSN 0008-6215. PMID 12681926.
  10. ^ a b c d e f g Langley, David B.; Harty, Derek W.S.; Jacques, Nicholas A.; Hunter, Neil; Guss, J. Mitchell; Collyer, Charles A. (March 2008). "Structure of N-acetyl-β-D-glucosaminidase (GcnA) from the Endocarditis Pathogen Streptococcus gordonii and its Complex with the Mechanism-based Inhibitor NAG-thiazoline". Journal of Molecular Biology. 377 (1): 104–116. doi:10.1016/j.jmb.2007.09.028. ISSN 0022-2836. PMID 18237743.
  11. ^ a b Ueda, Mitsuhiro; Arai, Motoo (January 1992). "Purification and Some Properties ofβ-N-Acetylglucosaminidase fromAeromonassp. 10S–24". Bioscience, Biotechnology, and Biochemistry. 56 (8): 1204–1207. doi:10.1271/bbb.56.1204. ISSN 0916-8451.
  12. ^ a b IWAMOTO, Tooru; INAOKA, Megumu; NAKA, Hideyuki (1983). "Mycelial .BETA.-N-acetylglucosaminidase of Streptomyces sp. having .BETA.-N-acetylgalactosaminidase activity". Agricultural and Biological Chemistry. 47 (8): 1913–1915. doi:10.1271/bbb1961.47.1913. ISSN 0002-1369.
  13. ^ VYAS, PRANAV; DESHPANDE, MUKUND V. (1989). "Chitinase production by Myrothecium verrucaria and its significance for fungal mycelia degradation". The Journal of General and Applied Microbiology. 35 (5): 343–350. doi:10.2323/jgam.35.343. ISSN 1349-8037.
  14. ^ a b c Miller, Morten; Palojärvi, Ansa; Rangger, Andrea; Reeslev, Morten; Kjøller, Annelise (February 1998). "The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil". Applied and Environmental Microbiology. 64 (2): 613–617. Bibcode:1998ApEnM..64..613M. doi:10.1128/aem.64.2.613-617.1998. ISSN 0099-2240. PMC 106091. PMID 9464399.
  15. ^ a b c Peters, G.; Saborowski, R.; Buchholz, F.; Mentlein, R. (1999-09-07). "Two distinct forms of the chitin-degrading enzyme N -acetyl-β- d -glucosaminidase in the Antarctic krill: specialists in digestion and moult". Marine Biology. 134 (4): 697–703. doi:10.1007/s002270050585. ISSN 0025-3162. S2CID 18922279.
  16. ^ a b Rollin, Marc; Coulaud, Romain; Rocher, Béatrice; Duflot, Aurélie; Poret, Agnes; Le Foll, Frank; Xuereb, Benoit (June 2021). "N-acetyl-β-d-glucosaminidase activity in Palaemon serratus - Methodological optimisation and intrinsic variability". Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 256: 110932. doi:10.1016/j.cbpa.2021.110932. ISSN 1095-6433. PMID 33677047. S2CID 232143454.
  17. ^ a b Jhaveri, Parth; Papastamatiou, Yannis P.; German, Donovan P. (November 2015). "Digestive enzyme activities in the guts of bonnethead sharks ( Sphyrna tiburo ) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts". Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 189: 76–83. doi:10.1016/j.cbpa.2015.07.013. hdl:10023/9230. ISSN 1095-6433. PMID 26239220.
  18. ^ a b Leigh, Samantha C.; Papastamatiou, Yannis P.; German, Donovan P. (2021-04-05). "Gut microbial diversity and digestive function of an omnivorous shark". Marine Biology. 168 (5). doi:10.1007/s00227-021-03866-3. ISSN 0025-3162. S2CID 233542070.
  19. ^ Lundblad, G.; Fänge, R.; Slettengren, K.; Lind, J. (August 1979). "Lysozyme, chitinase and exo-N-acetyl-?-D-glucosaminidase (NAGase) in lymphomyeloid tissue of marine fishes". Marine Biology. 53 (4): 311–315. doi:10.1007/bf00391613. ISSN 0025-3162. S2CID 85246855.
  20. ^ a b c d Calvo, P; Reglero, A; Cabezas, J A (1978-11-01). "Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller". Biochemical Journal. 175 (2): 743–750. doi:10.1042/bj1750743. ISSN 0264-6021. PMC 1186125. PMID 33660.
  21. ^ Nagai, Takeshi; Hamada, Moritsugu; Kai, Norihisa; Tanoue, Yasuhiro; Ueno, Shunshiro; Nagayama, Fumio (1997). "Existence of Chitinase and β-N-Acetylglucosaminidase in Several Jellyfish". Fisheries Science. 63 (1): 157–158. doi:10.2331/fishsci.63.157. ISSN 0919-9268. S2CID 83967485.
  22. ^ a b Cohen-Kupiec, Rachel; Chet, Ilan (June 1998). "The molecular biology of chitin digestion". Current Opinion in Biotechnology. 9 (3): 270–277. doi:10.1016/s0958-1669(98)80058-x. ISSN 0958-1669. PMID 9650272.
  23. ^ a b Ekenler, Mine; Tabatabai, M. A. (2004-12-31). "β-Glucosaminidase Activity as an Index of Nitrogen Mineralization in Soils". Communications in Soil Science and Plant Analysis. 35 (7–8): 1081–1094. doi:10.1081/css-120030588. ISSN 0010-3624. S2CID 95007714.
  24. ^ Çuhadar, Serap; Semerci, Tuna (2016). "Renal Biomarkers N-Acetyl-Beta-d-Glucosaminidase (NAG), Endothelin, and Their Application". Biomarkers in Kidney Disease. pp. 369–396. doi:10.1007/978-94-007-7699-9_8. ISBN 978-94-007-7698-2.
  25. ^ Tews, Ivo; Vincentelli, Renauld; Vorgias, Constantin E. (April 1996). "N-Acetylglucosaminidase (chitobiase) from Serratia marcescens: gene sequence, and protein production and purification in Escherichia coli". Gene. 170 (1): 63–67. doi:10.1016/0378-1119(95)00848-9. ISSN 0378-1119. PMID 8621090.
  26. ^ a b VORGIAS, C. E.; PERRAKIS, A.; TEWS, I. (October 1996). "Structure-Function Studies on the Chitinolytic Enzymes of Serratia marcescens Chitinase and Chitobiase". Annals of the New York Academy of Sciences. 799 (1 Enzyme Engine): 190–192. Bibcode:1996NYASA.799..190V. doi:10.1111/j.1749-6632.1996.tb33198.x. ISSN 0077-8923. PMID 8958090. S2CID 35850837.
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