Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes (original) (raw)
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.
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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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Authors and Affiliations
- Department of Physiology, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500, Copenhagen, Denmark
Ole Olsen & Karl Kristian Thomsen - Department of Food Technology, Microbiology Unit, Humboldt University, Warschauer Strasse 43, O-1017, Berlin, Federal Republic of Germany
Rainer Borriss & Ortwin Simon
Authors
- Ole Olsen
- Rainer Borriss
- Ortwin Simon
- Karl Kristian Thomsen
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Communicated by H. Böhme
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Olsen, O., Borriss, R., Simon, O. et al. Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes.Molec. Gen. Genet. 225, 177–185 (1991). https://doi.org/10.1007/BF00269845
- Received: 20 September 1990
- Issue date: February 1991
- DOI: https://doi.org/10.1007/BF00269845