1887

Abstract

A gain of function mutant of Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, D, grew in galactitol minimal medium with a growth rate of 0-11 h ( = 6-3 h). In crude extracts of D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass ( ) of the native GDH was 110000. SDS-PAGE resulted in one single band that represented a polypeptide with a of 28000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4-2. The enzyme was specific for NAD but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols. The apparent values were: galactitol, 240 mM; D-threitol, 85 mM; 1,2-hexandiol, 0-2 mM; NAD, 12μM; L-erythrulose, 144 mM; acetoin, 62 mM; dihydroxyacetone, 48 mM; and NADH, 4μM. GDH activity was strictly dependent on the presence of divalent cations. The properties of GDH are different to any of the three polyol dehydrogenases from Si4. In addition, comparison of the N-terminal amino acid sequence of the isolated GDH with the N-terminal sequence of the other three polyol dehydrogenases clearly demonstrates that GDH is an additional enzyme, so far unrecognized in the wild-type strain.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-8-1865
1995-08-01
2021-05-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/8/mic-141-8-1865.html?itemId=/content/journal/micro/10.1099/13500872-141-8-1865&mimeType=html&fmt=ahah

References

  1. Anderson LO., Borg H., amp;Michaelson M. 1972; Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity.. FEBS Lett 20:199–202
    [Google Scholar]
  2. Buchholz S.E., O’Mullan P., Eveleigh D.E. 1988; Growth of Zymomonas mobilis CP4 on mannitol.. Appl Microbiol Biotechnol 29:275–281
    [Google Scholar]
  3. Chen Y.M., Chakrabarti T., Lin E.C.C. 1984; Constitutive activation of L-fucose genes by an unlinked mutation in E. coli.. J Bacteriol 159:725–729
    [Google Scholar]
  4. Davis B.J. 1964; Disk electrophoresis. II. Method and application to human serum protein.. Ann NY Acad Sci 121:404–423
    [Google Scholar]
  5. Doten R.C., Mortlock R.P. 1984; Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniae.. J Bacteriol 159:730–735
    [Google Scholar]
  6. Drueckhammer D.G., Hennen W.J., Pederson R.L., Barbas C.F., Gautheron C.M., Krach T., Wong C.H. 1991; Enzyme catalysis in synthetic carbohydrate chemistry.. Synthesis 7:499–525
    [Google Scholar]
  7. Dykhuizen D.E., Haiti D.L. 1983; Selection in chemostats.. Microbiol Rev 47:150–168
    [Google Scholar]
  8. Goa J. 1953; A micro Biuret method for protein determination.. Scand J Clin Invest 5:218–222
    [Google Scholar]
  9. Hall B.G. 1981; Changes in the substrate specificities of an enzyme during directed evolution of new functions.. Biochemistry 20:4042–4049
    [Google Scholar]
  10. Hall B.G., Yokoyama S., Calhoun D.H. 1983; Role of cryptic genes in microbial evolution.. Mol Biol Evol 1:109–124
    [Google Scholar]
  11. Hartley B.S., Burleigh B.D., Midwinter G.G., Moore C.H., Morris H.R., Rigby P.W.J., Smith M.J., Taylor S.S. 1972; Where do enzymes come from?. FEBS Lett 29:151–176
    [Google Scholar]
  12. Hegeman G.D., Rosenberg S.L. 1970; The evolution of bacterial enzyme systems.. Anna Rev Microbiol 24:429–462
    [Google Scholar]
  13. Jeffery J., Jürnvall H. 1988; Sorbitol dehydrogenase.. Adv Enzymol Relat Areas Mol Biol 61:47–106
    [Google Scholar]
  14. Jeffery J., Chesters J., Mills C., Sadler P.J., Jürnvall H. 1984; Sorbitol dehydrogenase is a zinc enzyme.. EMBO J 3:357–360
    [Google Scholar]
  15. Kahle C., Schneider K.H., Giffhorn F. 1992; Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.. J Gen Microbiol 138:1277–1281
    [Google Scholar]
  16. Keinan E., Hafeli E.K., Seth K.K., Lamed R. 1986; Thermo-stable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii.. J Am Chem Soc 108:162–169
    [Google Scholar]
  17. Kondratieva E.N. 1979; Interrelation between modes of carbon assimilation and energy production in phototrophic purple and green bacteria.. Int Rev Biochem 21:117–175
    [Google Scholar]
  18. Kyslik P., Dobisova M. 1986; Selection of ribitol dehydrogenase hyperproducing strains in a chemostat culture of Escherichia coli 1EA at different dilution rates.. Biotechnol Lett 8:235–240
    [Google Scholar]
  19. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 227:680–685
    [Google Scholar]
  20. Lambert T., Ploy M.C., Courvalin P. 1994; A spontaneous point mutation in the aac(6’)-Ib’ gene results in altered substrate specificity of aminoglycoside 6’-N-acetyltransferase of a Pseudomonas fluorescens strain.. FEMS Microbiol Lett 115:297–304
    [Google Scholar]
  21. LeBlanc D.J., Mortlock R.P. 1971; Metabolism of D-arabinose: a new pathway in Escherichia coli.. J Bacteriol 106:90–96
    [Google Scholar]
  22. Lerner S.A., Wu T.T., Lin E.C.C. 1964; Evolution of a catabolic pathway in bacteria.. Science 146:1313–1315
    [Google Scholar]
  23. Mayers-KUntzer H., Reichert A., Schneider K.H., Giffhorn F. 1994; Isolation and characterization of a L-glucitol dehydrogenase from the newly isolated bacterium Pseudomonas sp. Ac.. J Biotechnol 36:157–164
    [Google Scholar]
  24. Merril C.R., Goldman D., Sedman S.A., Ebert M.H. 1981; Ultrasensitive stain for proteins in polyacrylamide gels shows regional variations in cerebrospinal fluid proteins.. Science 211:1437–1438
    [Google Scholar]
  25. Neuberger M.S., Patterson R.A., Hartley B.S. 1979; Puri-fication and properties of Klebsiella aerogenes D-arabitol dehydrogenase.. Biochem J 183:31–42
    [Google Scholar]
  26. Ng K., Ye R., Wu X.C., Wong S.L. 1992; Sorbitol dehydrogenase from Bacillus subtilis - purification, characterization and gene cloning.. J Biol Chem 267:24989–24994
    [Google Scholar]
  27. Parker L.L., Betts P.W., Hall B.G. 1988; Activation of a cryptic gene by excision of a DNA fragment.. J Bacteriol 170:218–222
    [Google Scholar]
  28. Pfennig N., Lippert K.D. 1966; Über das Vitamin B 12- Bediirfnis phototropher Schwefelbakterien.. Arch Microbiol 55:245–256
    [Google Scholar]
  29. Rigby P.W.J., Burleigh B.D. Jr Hartley B.S. 1974; Gene duplication in experimental enzyme evolution.. Nature 251:200–204
    [Google Scholar]
  30. Rode H., Giffhorn F. 1982; D-( - )-Tartrate dehydratase of Rhodopseudomonas sphaeroides: purification, characterization and application to enzymatic determination of D-( -)-tartrate.. J Bacteriol 150:1061–1068
    [Google Scholar]
  31. Rode H., Giffhorn F. 1983; Adaptation of Rhodopseudomonas sphaeroides to growth on D-( - )-tartrate and large-scale production of constitutive D-( -)-tartrate-dehydratase during growth on DL- malate.. Appl Environ Microbiol 45:716–719
    [Google Scholar]
  32. Schachmann H.K. 1959 Ultracentrifugation in Biochemistry. New York: Academic Press;
    [Google Scholar]
  33. Schauder S., Schneider K.H., Giffhorn F. 1995; Polyol metabolism of RPodobacter sphaeroides-. biochemical characterization of a short-chain sorbitol dehydrogenase.. Microbiology 141:1857–1863
    [Google Scholar]
  34. Schneider K.H., Giffhorn F. 1989; Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides.. Eur J Biochem 184:15–19
    [Google Scholar]
  35. Schneider K.H., Giffhorn F. 1991; Sorbitol dehydrogenase from Pseudomonas sp.: purification, characterization and application to quantitative determination of sorbitol.. Enzyme Microb Technol 13:332–337
    [Google Scholar]
  36. Schneider K.H., Giffhorn F., Kaplan S. 1993; Cloning, nucleotide sequence and characterization of the mannitol de-hydrogenase gene from Rhodobacter sphaeroides.. J Gen Microbiol 139:2475–2484
    [Google Scholar]
  37. Schwartz D., Stein M., Schneider K.H., Giffhorn F. 1994; Synthesis of D-xylulose from D-arabitol by enzymatic conversion with immobilized mannitol dehydrogenase from Rhodobacter sphaeroides.. J Biotechnol 33:95–101
    [Google Scholar]
  38. Srinivasan K., Konstantinidis A., Sinnott M.L., Hall B.G. 1993; Large changes of transition-state structure during ex-perimental evolution of an enzyme.. Biochem J 291:15–17
    [Google Scholar]
  39. Szymona M., Doudoroff M. 1960; Carbohydrate metabolism in Rhodopseudomonas sphaeroides.. J Gen Microbiol 22:167–183
    [Google Scholar]
  40. Taylor S.S., Rigby P.W.J., Hartley B.S. 1974; Ribitol dehydrogenase from Klebsiella aerogenes.Purification and subunit structure.. Biocbem J 141:693–700
    [Google Scholar]
  41. Wawzonek Thompson L., Krawiec S. 1983; Acquisitive-evolution of ribitol dehydrogenase in Klebsiella pneumoniae.. J Bacteriol 154:1027–1031
    [Google Scholar]
  42. Wojtkiewicz B., Szmidzinski R., Jezierska A., Cocito C. 1988; Identification of a salvage pathway for D-arabinose in Mycobacterium smegmatis.. Eur J Biochem 172:197–203
    [Google Scholar]
  43. Wu T.T., Lin E.C.C., Tanaka S. 1968; Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon source.. J Bacteriol 96:447–456
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-8-1865
Loading
/content/journal/micro/10.1099/13500872-141-8-1865
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error