Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2.

An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3+/-4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC-MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 degrees C in Tris/HCl buffer. It showed a half-life at 30 degrees C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 degrees C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a beta-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 gl(-1) and a vmax of 0.72 gl(-1)min(-1). The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.

[1]  Hans-Karl Rouette,et al.  Encyclopedia of Textile Finishing , 2001 .

[2]  A. Voragen,et al.  Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme , 2000, Applied and Environmental Microbiology.

[3]  I. Hardin,et al.  ENZYMATIC SCOURING OF COTTON : SURFACTANTS, AGITATION, AND SELECTION OF ENZYMES , 1998 .

[4]  S. Pérez,et al.  A complex plant cell wall polysaccharide: rhamnogalacturonan II. A structure in quest of a function. , 2003, Biochimie.

[5]  R. Vaughn,et al.  Purification and Properties of a Polygalacturonic Acid Trans-Eliminase Produced by Bacillus pumilus , 1971, Journal of bacteriology.

[6]  E. Vandamme,et al.  Pectin, pectinase and protopectinase: production, properties, and applications. , 1993, Advances in applied microbiology.

[7]  A. Karbassi,et al.  Purification and properties of polygalacturonic acid trans-eliminase from Bacillus stearothermophilus. , 1980, Canadian journal of microbiology.

[8]  Y. Hsieh,et al.  Enzymatic Scouring to Improve Cotton Fabric Wettability , 1998 .

[9]  S. Solís,et al.  Isolation of endopolygalacturonase hyperproducing mutants ofAspergillus sp. CH-Y-1043 , 1990, Biotechnology Letters.

[10]  O. Olsen,et al.  Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin. , 1995, Microbiology.

[11]  S. Pérez,et al.  The three-dimensional structures of the pectic polysaccharides , 2000 .

[12]  L. Paleg Citric Acid Interference in Estimation of Reducing Sugars with Alkaline Copper Reagents , 1959 .

[13]  Ronald M. Atlas,et al.  Handbook of microbiological media , 1993 .

[14]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  A. Wheals,et al.  Endopolygalacturonase secretion by Kluyveromyces marxianus and other cocoa pulp degrading yeasts , 1997 .

[16]  G. L. Miller Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[17]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[18]  K. Mechtler,et al.  Characterisation of the rubber elongation factor from ammoniated latex by electrophoresis and mass spectrometry. , 2000, Journal of chromatography. A.

[19]  M Takao,et al.  Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47 , 2000, Bioscience, biotechnology, and biochemistry.

[20]  J. Benen,et al.  Inversion of configuration during hydrolysis of α‐1,4‐galacturonidic linkage by three Aspergillus polygalacturonases , 1996, FEBS letters.

[21]  Marinus Warmoeskerken,et al.  A fast, continuous enzyme-based pretreatment process concept for cotton containing textiles , 2004 .

[22]  F. Brühlmann Purification and characterization of an extracellular pectate lyase from an Amycolata sp. , 1995, Applied and environmental microbiology.

[23]  Youliang Wang,et al.  Cloning and expression of a pectate lyase gene from Bacillus alcalophillus NTT33 , 2003 .

[24]  Y. Hakamada,et al.  Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate. , 1999, Bioscience, biotechnology, and biochemistry.

[25]  L. Ingram,et al.  Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16 , 1992, Journal of bacteriology.

[26]  B. Miller,et al.  Liquid porosimetry : new methodology and applications , 1994 .

[27]  J. Beeumen,et al.  Purification, characterisation and mode of action of an endo-polygalacturonase from the psychrophilic fungus Mucor flavus , 2003 .

[28]  Sridevi Annapurna Singh,et al.  Exopolygalacturonate lyase from a thermophilic Bacillus sp. , 1999 .

[29]  F. Stutzenberger Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca , 1987, Journal of bacteriology.

[30]  Tohru Kobayashi,et al.  A New High-alkaline and High-molecular-weight Pectate Lyase from a Bacillus Isolate: Enzymatic Properties and Cloning of the Gene for the Enzyme , 2000, Bioscience, biotechnology, and biochemistry.

[31]  N. Hugouvieux-Cotte-Pattat,et al.  Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors , 1997, Journal of bacteriology.

[32]  S. Ito,et al.  Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate. , 1999, Biochimica et biophysica acta.

[33]  J. Benen,et al.  Characterization of Aspergillus niger pectate lyase A. , 2000, Biochemistry.

[34]  M. Vršanská,et al.  Inverting character of α-glucuronidase A from Aspergillus tubingensis , 2000 .

[35]  G. Guebitz,et al.  Bio-preparation of cotton fabrics , 2001 .

[36]  A. Voragen,et al.  Analysis of partially methyl-esterified galacturonic acid oligomers by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1998, Analytical biochemistry.

[37]  J. Benen,et al.  Modes of Action of Five Different Endopectate Lyases from Erwinia chrysanthemi 3937 , 1999 .

[38]  M. Pizzigallo,et al.  Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp , 1997 .

[39]  T. Sakamoto,et al.  Purification, characterization, and production of two pectic transeliminases with protopectinase activity from Bacillus subtilis. , 1994, Bioscience, biotechnology, and biochemistry.

[40]  K. Sawada,et al.  Bioscouring of cotton with pectinase enzyme , 2008 .

[41]  R. Vaughn,et al.  Preparation, separation and degradation of oligouronides produced by the polygalacturonic acid transeliminase of Bacillus pumilus. , 1976, Journal of chromatography.

[42]  R. Linhardt,et al.  Conformational study of synthetic delta 4-uronate monosaccharides and glycosaminoglycan-derived disaccharides. , 1998, Carbohydrate research.