Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry.
An on-line system based on microdialysis sampling (MD), micro-high performance anion exchange chromatography (micro-HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on-line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5-mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro-HPAEC-IPED/MS and on-line desalting of hydrolysates enabled injection (5 microL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm-selectivity of the MD membrane (30 kDa cut-off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on-line with micro-HPAEC-IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co-elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean-up combined with on-line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.
Rumbold
Karl
K
Department of Microbiology, University of Stellenbosch, P/Bag X1, 7602 Matieland, South Africa.
Okatch
Harriet
H
Torto
Nelson
N
Siika-Aho
Matti
M
Gübitz
Georg
G
Robra
Karl-Heinz
KH
Prior
Bernard
B
D002480:Cellulase / Q000737:chemistry; D002482:Cellulose / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D004950:Esterases / Q000737:chemistry; D005052:Eucalyptus / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008031:Lignin / Q000737:chemistry*; D017551:Microdialysis / Q000379:methods; D010936:Plant Extracts / Q000737:chemistry; D011492:Protein Hydrolysates; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013997:Time Factors; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry
D010936:Plant Extracts; D011492:Protein Hydrolysates; C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; C027433:bagasse; D006867:Hydrolases; D004950:Esterases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase