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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
Article

Summary

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 

Abbreviations

AMY

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

MAC

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

SUB

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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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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