title: Overproduction of a thermostable 4-α-glucanotransferase by codon optimization at N-terminus region.
abstract: BACKGROUND
  4-α-Glucanotransferases are useful enzymes to modify starch owing to their transglycosylation activity. In this study, codon optimizations were conducted to overproduce a thermostable 4-α-glucanotransferase from Thermus thermophilus (TTαGT).
  
  
  RESULTS
  Two variants, termed TTαGT-P4CCG and TTαGT-mut6, were constructed, which have the optimized codon at the first rare codon and optimized codons at all six chosen rare codons at the N-terminus of TTαGT, respectively. In the Escherichia coli system, the expression of both optimized genes was enhanced by about 100-fold relative to that of the original gene, whereas all six mutated codons contributed to the overall enhancement of TTαGT production in Bacillus subtilis. On the basis of the αGTase activity of the crude cell extracts, relative activities of 1:2.9:5.8 were determined for TTαGT, TTαGT-P4CCG and TTαGT-mut6, respectively, in B. subtilis. In addition, the activity of TTαGT-mut6 from B. subtilis grown without antibiotics was as much as that with the antibiotics. Finally, after heat treatment, the specific activity of TTαGT-mut6 from B. subtilis was 1.5-fold greater than that from E. coli.
  
  
  CONCLUSIONS
  The codon-optimized TTαGT that was produced in a GRAS microorganism, B. subtilis, without the selection antibiotics is potentially useful in the food industry as a food-grade enzyme.
authors.affiliation: Department of Food and Biotechnology, Korea University, 2511 Sejong-Ro, Sejong, 339-700, Korea.
authors.forename: Min-Su
authors.initials: MS
authors.lastname: Kim
authors.forename: Jun-Hyuck
authors.initials: JH
authors.lastname: Jang
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006004:Glycogen Debranching Enzyme System / Q000145:classification* / Q000378:metabolism; D030781:Organisms, Genetically Modified; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D006004:Glycogen Debranching Enzyme System; C020569:4 alpha-glucanotransferase
keywords: 4-α-glucanotransferase; codon optimization; food-grade enzyme; overproduction