title: Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome.
abstract: The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.
authors: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors: Soumyadeep
authors: S
authors: Chakraborty
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Vania O
authors: VO
authors: Fernandes
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Fernando M V
authors: FM
authors: Dias
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Jose A M
authors: JA
authors: Prates
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Luis M A
authors: LM
authors: Ferreira
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Carlos M G A
authors: CM
authors: Fontes
authors: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors: Arun
authors: A
authors: Goyal
authors: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
authors: Maria S J
authors: MS
authors: Centeno
mesh_terms: D001483:Base Sequence; D002482:Cellulose / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D010368:Pectins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature
chemical_list: D017931:DNA Primers; D010368:Pectins; D002482:Cellulose; D011133:Polysaccharide-Lyases