Purification and Characterization of Extremely Thermostable Beta-mannanase, Beta-mannosidase, and Alpha-galactosidase from the Hyperthermophilic Eubacterium Thermotoga Neapolitana 5068

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 1997 Jan 1

PMID 8979350

Citations 28

Authors

G D Duffaud

C M McCutchen

P Leduc

K N Parker

R M Kelly

Affiliations

Soon will be listed here.

Abstract

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.

Citing Articles

Purification, Cloning, Functional Expression, Structure, and Characterization of a Thermostable β-Mannanase from B168 and Its Efficiency in Production of Mannooligosaccharides from Coffee Wastes.

Suzuki K, Michikawa M, Sato H, Yuki M, Kamino K, Ogasawara W J Appl Glycosci (1999). 2021; 65(2):13-21.

PMID: 34354508 PMC: 8056896. DOI: 10.5458/jag.jag.JAG-2017_018.

Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium.

Baudrexl M, Schwarz W, Zverlov V, Liebl W Sci Rep. 2019; 9(1):15924.

PMID: 31685873 PMC: 6828813. DOI: 10.1038/s41598-019-52251-0.

Technological Aspects of the Production of Fructo and Galacto-Oligosaccharides. Enzymatic Synthesis and Hydrolysis.

Martins G, Ureta M, Tymczyszyn E, Castilho P, Gomez-Zavaglia A Front Nutr. 2019; 6:78.

PMID: 31214595 PMC: 6554340. DOI: 10.3389/fnut.2019.00078.

Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications.

Aulitto M, Fusco S, Limauro D, Fiorentino G, Bartolucci S, Contursi P World J Microbiol Biotechnol. 2019; 35(2):32.

PMID: 30701316 DOI: 10.1007/s11274-019-2591-3.

Microbial Diversity in Extreme Marine Habitats and Their Biomolecules.

Poli A, Finore I, Romano I, Gioiello A, Lama L, Nicolaus B Microorganisms. 2017; 5(2).

PMID: 28509857 PMC: 5488096. DOI: 10.3390/microorganisms5020025.

References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

McCleary B . A simple assay procedure for beta-D-mannanase. Carbohydr Res. 1978; 67(1):213-21. DOI: 10.1016/s0008-6215(00)83743-x. View

Vieille C, Hess J, Kelly R, Zeikus J . xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol. 1995; 61(5):1867-75. PMC: 167449. DOI: 10.1128/aem.61.5.1867-1875.1995. View

Smith P, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M . Measurement of protein using bicinchoninic acid. Anal Biochem. 1985; 150(1):76-85. DOI: 10.1016/0003-2697(85)90442-7. View

Belkin S, Wirsen C, Jannasch H . A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl Environ Microbiol. 1986; 51(6):1180-5. PMC: 239042. DOI: 10.1128/aem.51.6.1180-1185.1986. View

Moore J, Markiewicz P, Miller J . Identification and sequencing of the Thermotoga maritima lacZ gene, part of a divergently transcribed operon. Gene. 1994; 147(1):101-6. DOI: 10.1016/0378-1119(94)90046-9. View

Hobbie J, Daley R, Jasper S . Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977; 33(5):1225-8. PMC: 170856. DOI: 10.1128/aem.33.5.1225-1228.1977. View

Ruttersmith L, Daniel R . Thermostable beta-glucosidase and beta-xylosidase from Thermotoga sp. strain FjSS3-B.1. Biochim Biophys Acta. 1993; 1156(2):167-72. DOI: 10.1016/0304-4165(93)90132-r. View

Zapater I, Ullah A, Wodzinski R . Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization. Prep Biochem. 1990; 20(3-4):263-96. DOI: 10.1080/00327489008050201. View

10.

SIMPSON H, Haufler U, Daniel R . An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga. Biochem J. 1991; 277 ( Pt 2):413-7. PMC: 1151249. DOI: 10.1042/bj2770413. View

11.

Ruttersmith L, Daniel R . Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Purification and properties. Biochem J. 1991; 277 ( Pt 3):887-90. PMC: 1151328. DOI: 10.1042/bj2770887. View

12.

McCleary B, Matheson N . Enzymic analysis of polysaccharide structure. Adv Carbohydr Chem Biochem. 1986; 44:147-276. DOI: 10.1016/s0065-2318(08)60079-7. View

13.

Adams M, Perler F, Kelly R . Extremozymes: expanding the limits of biocatalysis. Biotechnology (N Y). 1995; 13(7):662-8. DOI: 10.1038/nbt0795-662. View

14.

Liebl W, Feil R, Gabelsberger J, KELLERMANN J, Schleifer K . Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli. Eur J Biochem. 1992; 207(1):81-8. DOI: 10.1111/j.1432-1033.1992.tb17023.x. View

15.

Gherardini F, Salyers A . Characterization of an outer membrane mannanase from Bacteroides ovatus. J Bacteriol. 1987; 169(5):2031-7. PMC: 212081. DOI: 10.1128/jb.169.5.2031-2037.1987. View

16.

Talbot G, Sygusch J . Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus. Appl Environ Microbiol. 1990; 56(11):3505-10. PMC: 185002. DOI: 10.1128/aem.56.11.3505-3510.1990. View

17.

Dakhova O, Kurepina N, Zverlov V, Svetlichnyi V, Velikodvorskaya G . Cloning and expression in Escherichia coli of Thermotoga neapolitana genes coding for enzymes of carbohydrate substrate degradation. Biochem Biophys Res Commun. 1993; 194(3):1359-64. DOI: 10.1006/bbrc.1993.1974. View

18.

Winterhalter C, Liebl W . Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8. Appl Environ Microbiol. 1995; 61(5):1810-5. PMC: 1388439. DOI: 10.1128/aem.61.5.1810-1815.1995. View

19.

Rios S, Pedregosa A, Fernandez Monistrol I, Laborda F . Purification and molecular properties of an alpha-galactosidase synthesized and secreted by Aspergillus nidulans. FEMS Microbiol Lett. 1993; 112(1):35-41. DOI: 10.1111/j.1574-6968.1993.tb06420.x. View

20.

BAHL O, Agrawal K . Glycosidases of Aspergillus niger. I. Purification and characterization of alpha- and beta-galactosidases and beta-N-acetylglucosaminidase. J Biol Chem. 1969; 244(11):2970-8. View