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. 1994 Aug;60(8):2911–2915. doi: 10.1128/aem.60.8.2911-2915.1994

Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.

K Sakai 1, M Narihara 1, Y Kasama 1, M Wakayama 1, M Moriguchi 1
PMCID: PMC201742  PMID: 8085829

Abstract

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.

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Selected References

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  1. Dziadik-Turner C., Koga D., Mai M. S., Kramer K. J. Purification and characteristics of two beta-N-acetylhexosaminidases from the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae). Arch Biochem Biophys. 1981 Dec;212(2):546–560. doi: 10.1016/0003-9861(81)90398-2. [DOI] [PubMed] [Google Scholar]
  2. Koga K., Iwamoto Y., Sakamoto H., Hatano K., Sano M., Kato I. Purification and characterization of beta-N-acetylhexosaminidase from Trichoderma harzianum. Agric Biol Chem. 1991 Nov;55(11):2817–2823. [PubMed] [Google Scholar]
  3. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  4. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  5. Myerowitz R., Piekarz R., Neufeld E. F., Shows T. B., Suzuki K. Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7830–7834. doi: 10.1073/pnas.82.23.7830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Neote K., Bapat B., Dumbrille-Ross A., Troxel C., Schuster S. M., Mahuran D. J., Gravel R. A. Characterization of the human HEXB gene encoding lysosomal beta-hexosaminidase. Genomics. 1988 Nov;3(4):279–286. doi: 10.1016/0888-7543(88)90116-4. [DOI] [PubMed] [Google Scholar]
  7. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  8. REISSIG J. L., STORMINGER J. L., LELOIR L. F. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem. 1955 Dec;217(2):959–966. [PubMed] [Google Scholar]
  9. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  10. Somerville C. C., Colwell R. R. Sequence analysis of the beta-N-acetylhexosaminidase gene of Vibrio vulnificus: evidence for a common evolutionary origin of hexosaminidases. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6751–6755. doi: 10.1073/pnas.90.14.6751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Soto-Gil R. W., Zyskind J. W. N,N'-diacetylchitobiase of Vibrio harveyi. Primary structure, processing, and evolutionary relationships. J Biol Chem. 1989 Sep 5;264(25):14778–14783. [PubMed] [Google Scholar]
  12. Takayanagi T., Ajisaka K., Takiguchi Y., Shimahara K. Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochim Biophys Acta. 1991 Jul 12;1078(3):404–410. doi: 10.1016/0167-4838(91)90163-t. [DOI] [PubMed] [Google Scholar]
  13. Tsujibo H., Minoura K., Miyamoto K., Endo H., Moriwaki M., Inamori Y. Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Appl Environ Microbiol. 1993 Feb;59(2):620–622. doi: 10.1128/aem.59.2.620-622.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Usui T., Hayashi Y., Nanjo F., Ishido Y. Enzymatic synthesis of p-nitrophenyl N,N',N'',N'',N''''-pentaacetyl-beta-chitopentaoside in water-methanol system; significance as a substrate for lysozyme assay. Biochim Biophys Acta. 1988 Mar 23;953(2):179–184. [PubMed] [Google Scholar]
  15. Yamamoto K., Tsuji Y., Matsushita S., Kumagai H., Tochikura T. Purification and Properties of beta-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood. Appl Environ Microbiol. 1986 May;51(5):1019–1023. doi: 10.1128/aem.51.5.1019-1023.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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