Chitinase from Bacillus licheniformis DSM13: expression in Lactobacillus plantarum WCFS1 and biochemical characterisation.
The gene chi, coding for a GH18 chitinase from the Gram-positive bacterium Bacillus licheniformis DSM13 (ATCC 14580), was cloned into the inducible lactobacillal expression vectors pSIP403 and pSIP409, derived from the sakacin-P operon of Lactobacillus sakei, and expressed in the host strain Lactobacillus plantarum WCFS1. Both the complete chi gene including the original bacillal signal sequence as well as the mature chi gene were compared, however, no extracellular chitinase activity was detected with any of the constructs. The chitinase gene was expressed intracellularly as an active enzyme with these different systems, at levels of approximately 5mg of recombinant protein per litre of cultivation medium. Results obtained for the two different expression vectors that only differ in the promoter sequence were well comparable. To further verify the suitability of this expression system, recombinant, His-tagged chitinase Chi was purified from cell extracts of L. plantarum and characterised. The monomeric 65-kDa enzyme can degrade both chitin and chitosan, and shows properties that are very similar to those reported for the native chitinase purified from other B. licheniformis isolates. It shows good thermostability (half lives of stability of 20 and 8.4 days at 37 and 50°C, respectively), and good stability in the pH range of 5-10. The results presented lead the way to overproduction of chitinase in a food-grade system, which is of interest for the food and feed industry.
Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
Hoang Anh
HA
Nguyen
Thu-Ha
TH
Nguyen
Tien-Thanh
TT
Nguyen
Clemens K
CK
Peterbauer
Geir
G
Mathiesen
Dietmar
D
Haltrich
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D048271:Chitosan / Q000737:chemistry; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D048191:Lactiplantibacillus plantarum / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002686:Chitin; D048271:Chitosan; D002688:Chitinases