Molecular and General Genetics MGG

, Volume 225, Issue 2, pp 177–185 | Cite as

Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes

  • Ole Olsen
  • Rainer Borriss
  • Ortwin Simon
  • Karl Kristian Thomsen


Hybrid (1-3,1-4)-β-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-β-glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)-β-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-β-glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)-β-glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.

Key words

Hybrid enzymes Thermostability PCR Heterologous expression Mashing 



mature form of Bacillus amyloliquefaciens (1-3,1-4)-β-glucanase;


mature form of B. macerans (1-3,1-4)-β-glucanase


mature form of B. subtilis (1-3,1-4)-β-glucanase

H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M)

mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC


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  1. Alber T, Bell JA, Dao-Pin S, Nicolson H, Wozniak JA, Cook S, Matthews BW (1988) Replacement of Pro86 in phage T4 lysozyme extend an a-helix but do not alter protein stability. Science 239:631–635Google Scholar
  2. Anderson EH (1946) Growth requirement of virus-resistant mutants of Escherichia coli strain “B”. Proc Natl Acad Sci USA 32:120–128Google Scholar
  3. Anderson MA, Stone BA (1975) A new substrate for investigating the specificity of β-glucan hydrolases. FEBS Lett 52:502–507Google Scholar
  4. Borriss R (1981) Purification and isolation of an extracellular β-glucanase from Bacillus IMET 376 Z Allg Mikrobiol 21:7–17Google Scholar
  5. Borriss R, Zemek J (1981) β-1,3-1,4-glucanase in spore-forming microorganisms. Zentralbl Bakteriol II Abt 136:63–69Google Scholar
  6. Borriss R, Bäumlein H, Hofemeister J (1985) Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens. Appl Microbiol Biotechnol 22:63–71Google Scholar
  7. Borriss R, Manteuffel R, Hofemeister J (1988) Molecular cloning of a gene coding for thermostable β-glucanase from Bacillus macerans. J Basic Microbiol 28:3–10Google Scholar
  8. Borriss R, Olsen O, Thomsen KK, von Wettstein D (1989) Hybrid Bacillus endo-(1,3-1,4)-β-glucanases: Construction of recombinant genes and molecular properties of the gene products. Carlsberg Res Commun 54:41–54Google Scholar
  9. Borriss R, Büttner K, Mäntsälä P (1990) Structure of the β-1,3-1,4glucanase of Bacillus macerans: Homologies to other β-glucanases. Mol Gen Genet 222:278–283Google Scholar
  10. Bradford MM (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254Google Scholar
  11. Bryan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliand GL, Howard AJ, Poulos TL (1986) Proteases of enhanced stability. Characterization of a thermostable variant of subtilisin. Proteins 1:326–334Google Scholar
  12. Bueno A, Vazquez deAldana CR, Correra J, Villa TG, del Rey F (1990) Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-β-d-glucanase in Escherichia coli. J Bacteriol 172:2160–2167Google Scholar
  13. Cantwell BA, McConnell DJ (1983) Molecular cloning and expression of Bacillus subtilis β-glucanase gene in Escherichia coli. Gene 23:211–219Google Scholar
  14. Garnier J, Osguthorpe DJ, Robson B (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:97–120Google Scholar
  15. Hopp TP, Woods KR (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 78:3824–3828Google Scholar
  16. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77:61–68Google Scholar
  17. Imanaka T, Shibazaki M, Takagi M (1986) A new way of enhancing the thermostability of proteases. Nature 324:695–697Google Scholar
  18. Jørgensen KG (1988) Quantification of high molecular weight (1-3),(1-4)-β-d-glucan using calcofluor complex formation and flow injection analysis. I. Analytical principle and its standardization. Carlsberg Res Commun 53:277–285Google Scholar
  19. Jørgensen KG, Aastrup S (1988) Quantification of high molecular weight (1→3)(1→4)-β-d-glucan using calcofluor complex formation and flow injection analysis. II. Determination of total β-glucan content of barley malt. Carlsberg Res Commun 53:287–296Google Scholar
  20. Kaneko T, Song K-B, Hamamoto T, Kudo T, Horikoshi K (1989) Construction of a chimeric series of Bacillus cyclomaltodextrin glucanotransferases and analysis of the thermal stability and pH optima of the enzymes. J Gen Microbiol 135:3447–3457Google Scholar
  21. Matsumura M, Yasumura S, Aiba S (1986) Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. Nature 323:356–358Google Scholar
  22. Matthews BW, Nicholson H, Becktel WJ (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Nail Acad Sci USA 84:6663–6667Google Scholar
  23. McCleary BV (1988) Soluble, dye-labeled polysaccharides for the assay of endohydrolases. Methods Enzymol 160:74–86Google Scholar
  24. Mead DA, Szczwsna-Skorupa E, Kemper B (1986) Single-stranded DNA ‘blue’ T7-promoter plasmids: A versatile tandem promoter system for cloning and protein engineering. Protein Engineering 1:67–74Google Scholar
  25. Menèndez-Arias L, Argos P (1989) Engineering protein thermal stability. J Mol Biol 206:397–406Google Scholar
  26. Miller GL (1959) Use of dinitrosalicylic reagent for determination of reducing sugars. Anal Chem 31:426–428Google Scholar
  27. Olsen O and Thomsen KK (1990) Improvement of bacterial β-glucanase thermostability by glycosylation. J Gen Microbiol 137 (in press)Google Scholar
  28. Sauer RT, Hehir K, Stearman RS, Weiss MA, Jeiter-Nielsjson A, Suchanek EG, Pabo CO (1986) An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor. Biochemistry 25:5992–5999Google Scholar
  29. Yon J, Fried M (1989) Precise gene fusion by PCR. Nucleic Acids Res 17:4895Google Scholar
  30. Yuuki T, Tezuka H, Yabuuchi S (1989) Purification and some properties of two enzymes from a β-glucanase hyperproducing strain, Bacillus subtilis HL-25. Agric Biol Chem 53:2341–2346Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Ole Olsen
    • 1
  • Rainer Borriss
    • 2
  • Ortwin Simon
    • 2
  • Karl Kristian Thomsen
    • 1
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagenDenmark
  2. 2.Department of Food TechnologyMicrobiology Unit, Humboldt UniversityBerlinFederal Republic of Germany

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