title: Kytococcus sedentarius, the organism associated with pitted keratolysis, produces two keratin-degrading enzymes.
abstract: OBJECTIVE
  To determine characteristics of the extracellular enzyme activity of Kytococcus sedentarius on human callus.
  
  
  RESULTS
  A concentrate of a continuous culture supernatant fluid of K. sedentarius, which had callus-degrading activity, was subjected to a series of chromatographic purification procedures. The enzyme activity was found to be attributable to two proteases. These were capable of degrading both native callus and extracted keratin polypeptides and were purified to homogeneity, as shown by SDS-PAGE with silver staining. The enzymes P1 and P2 were 30 kDa and 50 kDa in size with isoelectric points of 4.6 and 2.7, respectively. The optimum conditions for callus-degrading activity were 40 degrees C, pH 7.1 for P1 and 50 degrees C, pH 7.5 for P2. P2 displayed increased activity in the presence of 800 mmol l(-1) NaCl and both enzymes were inhibited by PMSF (1 mmol(-1) Phenylmethylsulphoryl fluoride) and 1 mmol l(-1) EDTA. The main enzyme cleavage sites were Lys-Trp, Val-Lys, Gly-Asp and Asp-Arg, as determined after incubation of P1 and P2 with the beta-chain of insulin.
  
  
  CONCLUSIONS
  K. sedentarius produces two extracellular enzymes that independently degrade natural, insoluble human callus. Both enzymes are serine proteases and have cleavage preference sites that are present in a range of human keratins.
  
  
  CONCLUSIONS
  The identification, in K. sedentarius cultures, of two enzymes which can degrade human callus strengthens the hypothesis that this organism is responsible for the pitting in human epidermis observed in pitted keratolysis. These enzymes may be of commercial use in the biodegradation of a range of keratin polymers, biological washing powders and in the treatment of unwanted callus on human skin.
authors.affiliation: Skin Research Centre, Division of Microbiology, School of BMB, University of Leeds, Leeds, UK.
authors.forename: C M
authors.initials: CM
authors.lastname: Longshaw
authors.forename: J D
authors.initials: JD
authors.lastname: Wright
authors.forename: A M
authors.initials: AM
authors.lastname: Farrell
authors.forename: K T
authors.initials: KT
authors.lastname: Holland
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000502:physiology; D010450:Endopeptidases / Q000032:analysis / Q000145:classification / Q000302:isolation & purification*; D005110:Extracellular Space / Q000201:enzymology / Q000378:metabolism; D005533:Foot Dermatoses / Q000382:microbiology* / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007633:Keratins / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D013696:Temperature
chemical_list: D007328:Insulin; D010455:Peptides; D007633:Keratins; D010450:Endopeptidases