Characterization of water-soluble hemicelluloses from spruce and aspen employing SEC/MALDI mass spectroscopy

Partly depolymerized hemicelluloses isolated from wood chips of spruce and aspen employing microwave treatment were resolved using size-exclusion chromatography (SEC) into oligo- and polysaccharide fractions containing components with a narrow range of sizes, as determined by MALDI mass spectroscopy. The degree of substitution with acetyl moieties (DS) was also calculated on the basis of the MALDI-MS spectra obtained prior to and following deacetylation. For spruce hemicelluloses, the low molecular mass fraction contained small arabino-4-O-methylglucuronoxylan oligosaccharides, with DP values ranging from 4 to ~20, separated primarily on the basis of their charge density. The fraction eluted last consisted of an O-acetyl-(galacto)glucomannan polysaccharide of peak-average DP value (DPp) 14. The degree of substitution with acetyl groups (DS) decreased with decreasing DP, a value DS of 0.39 being obtained for the fraction with DPp 12. For the aspen hemicelluloses, the SEC fractions eluted first contained an acidic O-acetyl-4-O-methylglucuronoxylan polysaccharide with DP ranging from 10 to ~28 and an average DS of ~0.75. The fractions eluted last consisted of oligosaccharide mixtures composed primarily of small neutral O-acetyl-xylooligosaccharides (DPp 6, DS 0.41), together with minor quantities of an O-acetyl-glucomannan

Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose

Monocot residues such as corn stover and straw are often not fully exploited and constitute a potential substrate for bioethanol production. However, a number of factors such as high enzyme loadings make large-scale utilization economically difficult. Addition of non-ionic surfactants and poly(ethylene glycol) to enzymatic hydrolysis of various lignocellulosic substrates has been found to increase the conversion of cellulose into soluble, fermentable sugars. We have shown that surfactants are able to increase cellulose conversion with up to 70%. This provides an opportunity of decreasing enzyme loading while retaining the same degree of hydrolysis. Investigations of five wheat straw substrates produced with different pretreatment methods revealed that surfactants have a more pronounced effect on acid and steam treated straw than, e.g. ammonia and hydrogen peroxide treated straw. Thus, lignin content is not directly proportional with the potential surfactant effect. Studies of adsorption of cellulases support the theory that the main mechanism behind the surfactant effect is prevention of unspecific adsorption of enzyme on the substrate lignin. This is believed to be due to hydrophobic interaction between lignin and the surfactant, causing steric repulsion of enzyme from the lignin surface. More research is needed to reveal which factors influence enzyme and surfactant adsorption. (c) 2006 Elsevier Inc. All rights reserved

Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc2Man5 was identified as the predominant N-glycan, although lesser amounts of GlcNAc2Man7 and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations

Linear Dichroism of Chloroplasts and Subchloroplast Fractions Oriented by Flow

Chloroplasts as well as subchloroplast vesicles show linear dichroism (LD) when subjected to a hydrodynamic gradient. LD is positive for the ππ in-plane polarized bands indicating that the chlorophyll porphyrin ring is in average oriented parallel1 to the elongated membrane surfaces. The vesicle fraction enriched inphotosystem I1 shows lower LD/A at 660 nm indicating lower average orientation of chlorophyll and the fraction enriched in photosystem I shows higher LD/A at 700 nm indicating that P700 is more oriented than the bulk chlorophyll