Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS–PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca2+ and was stable up to 60 °C with 150 mM Ca2+. Another identical gene was located in tandem in the upstream of est119.

[1]  U. P. Fringeli,et al.  Activation of acetylcholinesterase by monovalent (Na+,K+) and divalent (Ca2+,Mg2+) cations. , 1984, Biochemistry.

[2]  S. Kanaya,et al.  Ca2+‐induced folding of a family I.3 lipase with repetitive Ca2+ binding motifs at the C‐terminus , 2001, FEBS letters.

[3]  Yuzuru Suzuki,et al.  A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability , 1991, Applied Microbiology and Biotechnology.

[4]  Gabriela Alves Macedo,et al.  Cutinases: properties and industrial applications. , 2009, Advances in applied microbiology.

[5]  W. Deckwer,et al.  Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli , 2006, Bioprocess and biosystems engineering.

[6]  H. Maeda,et al.  Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae , 2005, Applied Microbiology and Biotechnology.

[7]  Jian Chen,et al.  Identification and Characterization of Bacterial Cutinase* , 2008, Journal of Biological Chemistry.

[8]  W. Minor,et al.  Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution. , 1998, Structure.

[9]  Wei-Jye Shu,et al.  Degradation of polyethylene succinate (PES) by a new thermophilic Microbispora strain , 2007, Biodegradation.

[10]  K. Jaeger,et al.  Bacterial lipolytic enzymes: classification and properties. , 1999, The Biochemical journal.

[11]  T. Tan,et al.  Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution , 2006 .

[12]  W. Deckwer,et al.  Characterization of a new extracellular hydrolase from Thermobifida fusca degrading aliphatic-aromatic copolyesters. , 2005, Biomacromolecules.

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

[14]  Degradation of a Terephthalate-containing Polyester by Thermophilic Actinomycetes and Bacillus Species Derived from Composts , 2008 .

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  R C Cox,et al.  Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants. , 1999, Biochemistry.

[17]  Gabriela Alves Macedo,et al.  Chapter 4 Cutinases , 2009 .

[18]  Y. Tokiwa,et al.  Microbial degradation of poly(d-3-hydroxybutyrate) by a new thermophilic Streptomyces isolate , 2004, Biotechnology Letters.

[19]  W. Deckwer,et al.  Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium , 2007, Biotechnology and bioengineering.

[20]  W. Deckwer,et al.  Biodegradation of Aliphatic-Aromatic Copolyesters by Thermomonospora fusca and Other Thermophilic Compost Isolates , 1998, Applied and Environmental Microbiology.

[21]  I. Noda,et al.  Microbial degradation and physico-chemical alteration of polyhydroxyalkanoates by a thermophilic Streptomyces sp. , 2009, Biologia.

[22]  Zunsheng Wang,et al.  Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum. , 2006, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[23]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[24]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[25]  M. Nardini,et al.  α/β Hydrolase fold enzymes : the family keeps growing , 1999 .

[26]  T. Srikhirin,et al.  Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance , 2010, Applied Microbiology and Biotechnology.

[27]  Joji M. Otaki,et al.  Parallel and Antiparallel β-Strands Differ in Amino Acid Composition and Availability of Short Constituent Sequences , 2011, J. Chem. Inf. Model..

[28]  C. Hoogland,et al.  In The Proteomics Protocols Handbook , 2005 .

[29]  C. Vieille,et al.  Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability , 2001, Microbiology and Molecular Biology Reviews.

[30]  Maurizio Tosin,et al.  Activated Vermiculite, a Solid Bed for Testing Biodegradability under Composting Conditions , 1999 .

[31]  M. Aires-Barros,et al.  Cutinase: from molecular level to bioprocess development. , 1999, Biotechnology and bioengineering.

[32]  F. Kawai.  The Biochemistry and Molecular Biology of Xenobiotic Polymer Degradation by Microorganisms , 2010, Bioscience, biotechnology, and biochemistry.

[33]  L. Tighzert,et al.  An inert solid medium for simulation of material biodegradation in compost and achievement of carbon balance , 2004 .

[34]  Joel L. Sussman,et al.  The α/β hydrolase fold , 1992 .

[35]  Natalia Ivanova,et al.  Genome Sequence and Analysis of the Soil Cellulolytic Actinomycete Thermobifida fusca YX , 2007, Journal of bacteriology.

[36]  H. Nakajima,et al.  Different enantioselectivity of two types of poly(lactic acid) depolymerases toward poly(l-lactic acid) and poly(d-lactic acid) , 2011 .

[37]  Xiaoping Hu,et al.  Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119 , 2010, Applied Microbiology and Biotechnology.

[38]  Jung-Kee Lee,et al.  Thermostable Lipase of Bacillus stearothermophilus: High-level Production, Purification, and Calcium-dependent Thermostability , 2000, Bioscience, biotechnology, and biochemistry.

[39]  John M. Walker,et al.  The Proteomics Protocols Handbook , 2005, Humana Press.

[40]  U. Bornscheuer.  Microbial carboxyl esterases: classification, properties and application in biocatalysis. , 2002, FEMS microbiology reviews.