Optimization Of Expression Conditions Of The Acetylesterase CE16 From Hypocrea Jecorina Encoded By A Synthetic Gene And Expressed In Escherichia coli Cells

Open access

Abstract

Acetylesterase CE16 was identified as a part of the enzymatic cocktail secreted by fungus Hypocrea jecorina (anamorph: Trichoderma reesei) during its growth on cellulose. Later it was classified as the first member of a newly organized carbohydrate esterase family CE16. Further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared. The enzyme was composed of 348 amino acid residues from which the 1 - 19 formed a secretion signal peptide. Determined molecular mass of purified recombinant acetylesterase (Aes1) was 45 kDa which was more than molecular mass calculated from amino acid sequence. As it has been proved later, the difference was caused by the enzyme glycosylation. Glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. To make the future X-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. For these purposes, a synthetic gene optimized for protein expression in Escherichia coli was designed and synthetized. The first nonglycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells was mostly insoluble or aggregated. Conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. Presently, after optimization of all mentioned steps, the non-glycosylated recombinant CE16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • ADAV S.S. RAVINDRAN A. CHAO L.T. TAN L. SINGH S. SZE S.K.: Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei. J. Proteome Res. 10 2011 4579 - 4596.

  • BASHIRI S. VIKSTROM D. ISMAIL N.: Optimization of protein expression in Escherichia Coli. Biopharm. J. 28 2015 42 - 44.

  • BIELY P. CZISZÁROVÁ M. AGGER J.W. LI X.L. PUCHART V. VRŠANSKÁ M. EIJSINK V.G. WESTERENG B.: Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose. Biochim. Biophys. Acta 1840 2014 516 - 525.

  • BIELY P. MASTIHUBOVÁ M. TENKANEN M. EYZAGUIRRE J. LI X.L. VRŠANSKÁ M.: Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of β-D-xylopyranose and α-L-arabinofuranose. J. Biotechnol. 151 2011 137 - 142.

  • BIELY P. PULS J. SCHNEIDER H.: Acetyl xylan esterases in fungal cellulolytic systems. FEBS Letters 186 1985 80 - 84.

  • BUKAU B. HORWICH A.L.: The Hsp70 and Hsp60 chaperone machines. Cell J. 92 1998 351 - 366.

  • BURGESS R.R. MURRAY P.D.: Guide to protein purification. Elsevier Inc. San Diego 2009 851 pp.

  • CAFFALL K.H. MOHNEN D.: The structure function and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr. Res. 344 2009 1879 - 1900.

  • CARPITA N.C. GIBEAUT D.M.: Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3 1993 1 - 30.

  • FROGER A. HALL J.E.: Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. J. Vis. Exp. 6 2007 http://www.jove.com/index/Details.stp?ID=253 DOI: 10.3791/253.

  • GROTE A. HILLER K. SCHEER M. MUNCH R. NORTEMANN B. HEMPEL D.C. JAHN D.: JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 1 2005 526-531.

  • HANDRICK J.P. HARTL F.U.: The role of molecular chaperones in protein folding. FASEB J. 9 1995 1559 - 1569.

  • JONASSON P. LILJEQVIST S. NYGREN P. STAHL S.: Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem. 35 2002 91–105.

  • KEMPF B. BREMER E.: Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol. 170 1998 319 - 330.

  • KIRLEY T.L.: Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal. Biochem. 180 1989 231 - 236.

  • KOUTANIEMI S. VAN-GOOL M. P. JUVONENA M. JOKELAC J. HINZ S. W. SCHOLS H. A. TENKANENA M.: Distinct roles of carbohydrate esterase family CE16 acetylesterases and polymer-acting acetyl xylan esterases in xylan deacetylation. J. Biotechnol. 168 2013 684 - 692.

  • KREMNICKÝ L. BIELY P.: Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetylesterase. J. Mol. Catal. B: Enzym. 37 2005 72 - 78.

  • KREMNICKÝ L. MASTIHUBA V. COTE G.L.: Trichoderma reesei acetylesterase catalyzes transesterification in water. J. Mol. Catal. B: Enzym. 30 2004 229 - 239.

  • LAEMMLI U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 1970 680 - 685.

  • LI X.L. SKORY C.D. COTTA M.A. PUCHART V. BIELY P.: Novel family of carbohydrate esterases based on identification of the Hypocrea jecorina acetylesterase gene. Appl. Environ. Microbiol. 74 2008 7482 - 7489.

  • LOMBARD V. GOLACONDA R.H. DRULA E. COUTINHO P.M HENRISSAT B.: The Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42 2014 490 - 495.

  • MANIATIS T. FRITSCH E.F. SAMBROOK J.: Molecular cloning: a laboratory manual. Cold Spring Harbor N.Y.: Cold Spring Harbor Laboratory 1982 545 pp.

  • NAGATA S. SASAKI H. OSHIMA A. TAKEDA S. HASHIMOTO Y. ISHIDA A.: Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity. Biosci. Biotechnol. Biochem. 69 2005 740 - 746.

  • NITSCH CH. HEITLAND H.J. MARSEN H. SCHLÜUSSLER H.J.: Ullmann’s Encyclopedia of Industrial Chemistry Wiley-VCH Düsseldorf 2003 3008 pp.

  • OGANESYAN N. ANKOUDINOVA I. KIM S.H. KIM R.: Effect of Osmotic Stress and Heat Shock in Recombinant Protein Overexpression and Crystallization. Protein Expr. Purif. 52 2007 280 - 285.

  • POUTANEN K. SUNDBERG M.: An acetylesterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol. 28 1988 419 - 424.

  • PUCHART V. BERRIN J.G. HAON M. BIELY P.: A unique acetylesterase from Podospora anserina active on polymeric xylan. Appl. Microbiol. Biotechnol. 2015 1 - 12.

  • SASAKI H. ISHIDA A. HASHIMOTO Y. TAKEDA S. OSHIMA A. KAWAI H. NAGATA S.: Utilization of proline in Escherichia coli K-12 at different osmolarities. J. Biol. Sci. 6 2006 675 - 679.

  • SASAKI H. KURIKI K. OSHIMA A. ISHIDA A. NAGATA S.: Effect of overexpression of proline transporter ProP on high saline adaptation in Escherichia coli. Bull. Soc. Sea Water Sci. Jpn. 66 2012 30 - 35.

  • SIEVERS F. WILM A. DINEEN D. GIBSON T.J. KARPLUS K. LI W. LOPEZ R. MCWILLIAM H. REMMERT M. SÖDING J. THOMPSON J.D. HIGGINS D.G.: Fast scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7 2011 539.

  • SORENSEN H.P. MORTENSEN K.K.: Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb. Cell Fact. 4 2005 1 - 8.

  • WOOD R.W. LOOMIS A.L.: The physical and biological effects of high frequency sound waves of great intensity. Phil. Mag. 74 1927 414 - 436.

Search
Journal information
Impact Factor


CiteScore 2018: 0.68

SCImago Journal Rank (SJR) 2018: 0.173
Source Normalized Impact per Paper (SNIP) 2018: 0.288


Metrics
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 207 100 7
PDF Downloads 115 77 5