title: Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce.
abstract: BACKGROUND
  The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs).
  
  
  RESULTS
  LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis.
  
  
  CONCLUSIONS
  LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.
authors: Department of Food and Environmental Sciences, University of Helsinki, PO Box 27, Helsinki, 00014 Finland.
authors: Ulla
authors: U
authors: Moilanen
authors: Department of Food and Environmental Sciences, University of Helsinki, PO Box 27, Helsinki, 00014 Finland ; VTT Technical Research Centre of Finland, PO Box 1000, Espoo, 02044 Finland.
authors: Miriam
authors: M
authors: Kellock
authors: Department of Food and Environmental Sciences, University of Helsinki, PO Box 27, Helsinki, 00014 Finland ; Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, Aas, N-1432 Norway.
authors: Anikó
authors: A
authors: Várnai
authors: VTT Technical Research Centre of Finland, PO Box 1000, Espoo, 02044 Finland.
authors: Martina
authors: M
authors: Andberg
authors: Department of Food and Environmental Sciences, University of Helsinki, PO Box 27, Helsinki, 00014 Finland.
authors: Liisa
authors: L
authors: Viikari
keywords: Cellulose oxidation; Enzymatic hydrolysis; Laccase; Lignin; Mediator; Spruce
references: Biochem J. 1988 Sep 15;254(3):877-83
references: 3196301
references: J Biotechnol. 2006 Sep 1;125(2):198-209
references: 16621087
references: Biotechnol Bioeng. 1990 Aug 20;36(5):446-52
references: 18595100
references: Science. 2007 Feb 9;315(5813):804-7
references: 17289988
references: Biomacromolecules. 2004 Sep-Oct;5(5):1983-9
references: 15360314
references: FEBS Lett. 1990 Jul 2;267(1):99-102
references: 2365094
references: Biotechnol Bioeng. 2004 Jun 5;86(5):550-7
references: 15129438
references: Enzyme Microb Technol. 2011 Dec 10;49(6-7):492-8
references: 22142723
references: Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):6300-5
references: 21444820
references: Crit Rev Biotechnol. 2006 Oct-Dec;26(4):201-21
references: 17095432
references: Chem Rev. 1996 Nov 7;96(7):2563-2606
references: 11848837
references: Appl Biochem Biotechnol. 2010 May;161(1-8):347-64
references: 19898963
references: Biotechnol Adv. 2010 Nov-Dec;28(6):694-705
references: 20471466
references: Biotechnol Bioeng. 2010 Apr 1;105(5):871-9
references: 19998278
references: Enzyme Microb Technol. 2011 Mar 7;48(3):195-208
references: 22112901
references: Biochim Biophys Acta. 1998 Mar 2;1379(3):381-90
references: 9545600
references: Appl Environ Microbiol. 2000 Feb;66(2):524-8
references: 10653713
references: Appl Environ Microbiol. 1997 Dec;63(12):4627-32
references: 16535747
references: Org Biomol Chem. 2008 Mar 7;6(5):868-78
references: 18292878
references: J Biotechnol. 2004 Jan 8;107(1):65-72
references: 14687972
references: J Biol Chem. 1951 Nov;193(1):265-75
references: 14907713
references: Enzyme Microb Technol. 2013 Oct 10;53(5):315-21
references: 24034430
references: Biotechnol Bioeng. 2011 Dec;108(12):2823-34
references: 21702025
references: Appl Environ Microbiol. 2005 Apr;71(4):1775-84
references: 15812000
references: Eur J Biochem. 2002 Nov;269(21):5330-5
references: 12392567
references: Biotechnol Biofuels. 2012 Jul 02;5(1):45
references: 22747961
references: Science. 2004 Dec 24;306(5705):2206-11
references: 15618507
references: Bioresour Technol. 2011 Jan;102(2):1220-7
references: 20736135
references: Biotechnol Biofuels. 2014 Jan 08;7(1):6
references: 24401177
references: Bioresour Technol. 2012 Sep;119:114-22
references: 22728191