title: Construction of a chimeric thermoacidophilic beta-endoglucanase.
abstract: BACKGROUND
  The archeaon Sulfolobus solfataricus P2 encodes a thermoacidophilic cellulase which shows an extreme acid and thermal stability with a pH optimum at 1.8 and a temperature optimum at 80°C. This extraordinary enzyme could be useful for biotechnological exploitation but the expression and purification in expression hosts like E. coli is unsatisfactory due to the high aggregation tendency of the recombinant enzyme. The thermophilic cellulase CelA from Thermotoga maritima belongs to the same glycoside hydrolase family (GH12) but has a neutral pH optimum. In contrast to SSO1949 this enzyme is expressed partially soluble in E. coli.
  
  
  RESULTS
  We aimed to constructed a hybrid enzyme based on these two beta-endoglucanases which should successfully combine the advantageous properties of both cellulases, i.e. recombinant expression in E. coli, acidophily and thermophily. We constructed two hybrid proteins after bioinformatic analysis: both hybrids are expressed insoluble in E. coli, but one hybrid enzyme was successfully refolded from washed inclusion bodies.
  
  
  CONCLUSIONS
  The refolded active chimeric enzyme shows a temperature optimum of approximately 85°C and a pH optimum of approximately pH 3 thus retaining the advantageous properties of the Sulfolobus parent enzyme. This study suggests that the targeted construction of chimeric enzymes is an alternative to point mutational engineering efforts as long as parent enzymes with the wanted properties are available.
authors.affiliation: University of Applied Research of Northwestern Switzerland, Gründenstrasse 40, Muttenz, 4132, Switzerland.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kufner
authors.forename: Georg
authors.initials: G
authors.lastname: Lipps
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D011993:Recombinant Fusion Proteins; D002480:Cellulase