Cloning, sequence analysis, and characterization of a novel beta-glucosidase-like activity from Pichia etchellsii.
Genomic DNA fragment encoding a novel beta-glucosidase-like activity of the yeast Pichia etchellsii was cloned and expressed in Escherichia coli. An open-reading frame of 1515bp, termed mugA, coding for a protein of predicted molecular mass of approximately 54kDa was confirmed for this activity. The sequence of the deduced protein did not show homology with the generic beta-glucosidases but a high degree of identity was seen with several Ser-Asp (SD)-rich cell-surface-associated proteins. The secondary structure prediction program 3D-PSSM indicated the protein to be composed of largely helical and coiled structures, which was confirmed by circular dichroism spectroscopy. The encoded protein, MUGA, was purified by about 53-fold and characterized as a monomer of 52.1kDa by SDS-PAGE and MALDI-TOF. The protein displayed high hydrolytic activity on methylumbelliferyl beta-d-glucoside but relatively very little hydrolysis of p-nitrophenyl beta-d-glucoside and gentiobiose, characteristic substrates for beta-glucosidases. The binding experiments performed between P. etchellsii cells and the purified E. coli expressed MUGA indicated binding with the cell surface, which was monitored by fluorescence microscopy. In competition experiments with the SD dipeptide, less protein was shown to bind to the cell surface, in a concentration-dependent manner.
Roy
Pranita
P
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz-Khas, New Delhi 110016, India.
Mishra
Saroj
S
Chaudhuri
Tapan K
TK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010843:Pichia / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D001617:beta-Glucosidase