The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus.
A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.
Ramchuran
Santosh O
SO
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Sweden. santosh.ramchuran@biotek.lu.se
Mateus
Bruno
B
Holst
Olle
O
Karlsson
Eva Nordberg
EN
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology* / Q000235:genetics*; D020539:Sequence Analysis, Protein; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases