Effect of lignin-blocking agent on enzyme hydrolysis of acid pretreated hemp waste.

Hemp wastes (stems and branches), fractionated after hemp flower extraction for the production of cannabidiol oil, were utilized as a potentially renewable resource for the sugar flatform process. Hydrolysis of cellulose from the acid pretreated hemp biomass using a commercial enzyme was tested and evaluated for its chemical composition, morphological change, and sugar recovery. Acid pretreated hemp stems and branches, containing 1% glucan (w/v) solids, were hydrolyzed for 72 h using 25 mg enzyme protein per g glucan. A 54% glucose conversion was achieved from the treated branches versus a 71% yield from the treated stems. Raw branches and stems yielded 35% and 38% glucose, respectively. Further tests with a lignin-blocking additive (e.g. bovine serum albumin) resulted in a 72% glucose yield increase for stem hydrolysis using 10 mg enzyme protein per g glucan. While pretreatment promotes amorphous hemicellulose decrease and cellulose decomposition, it causes enzyme inhibition/deactivation due to potential inhibitors (phenols and lignin-derived compounds). This study confirms the addition of non-catalytic proteins enhances the cellulose conversion by avoiding non-productive binding of enzymes to the lignin and lignin-derived molecules, with lignin content determining the degree of inhibition and conversion efficiency.

Effect of Lignin Content on Cellulolytic Saccharification of Liquid Hot Water Pretreated Sugarcane Bagasse.

Lignin contributes to the rigid structure of the plant cell wall and is partially responsible for the recalcitrance of lignocellulosic materials to enzymatic digestion. Overcoming this recalcitrance is one the most critical issues in a sugar-flat form process. This study addresses the effect of low lignin sugarcane bagasse on enzymatic hydrolysis after liquid hot water pretreatment at 190 °C and 20 min (severity factor: 3.95). The hydrolysis of bagasse from a sugarcane line selected for a relatively low lignin content, gave an 89.7% yield of cellulose conversion to glucose at 40 FPU/g glucan versus a 68.3% yield from a comparably treated bagasse from the high lignin bred line. A lower enzyme loading of 5 FPU/g glucan (equivalent to 3.2 FPU/g total solids) resulted in 31.4% and 21.9% conversion yields, respectively, for low and high lignin samples, suggesting the significance of lignin content in the saccharification process. Further increases in the enzymatic conversion of cellulose to glucose were achieved when the bagasse sample was pre-incubated with a lignin blocking agent, e.g., bovine serum albumin (50 mg BSA/g glucan) at 50 °C for 1 h prior to an actual saccharification. In this work, we have demonstrated that even relatively small differences in lignin content can result in considerably increased sugar production, which supports the dissimilarity of bagasse lignin content and its effects on cellulose digestibility. The increased glucose yields with the addition of BSA helped to decrease the inhibition of non-productive absorption of cellulose enzymes onto lignin and solid residual lignin fractions.

Thermal Stabilization of Erwinia chrysanthemi pectin methylesterase a for application in a sugar beet pulp biorefinery.

Directed evolution approaches were used to construct a thermally stabilized variant of Erwinia chrysanthemi pectin methylesterase A. The final evolved enzyme has four amino acid substitutions that together confer a T(m) value that is approximately 11 degrees C greater than that of the wild-type enzyme, while maintaining near-wild-type kinetic properties. The specific activity, with saturating substrate, of the thermally stabilized enzyme is greater than that of the wild-type enzyme when both are operating at their respective optimal temperatures, 60 degrees C and 50 degrees C. The engineered enzyme may be useful for saccharification of biomass, such as sugar beet pulp, with relatively high pectin content. In particular, the engineered enzyme is able to function in biomass up to temperatures of 65 degrees C without significant loss of activity. Specifically, the thermally stabilized enzyme facilitates the saccharification of sugar beet pulp by the commercial pectinase preparation Pectinex Ultra SPL. Added pectin methylesterase increases the initial rate of sugar production by approximately 50%.

Chromosomal protein HMGN1 enhances the rate of DNA repair in chromatin.

We report that HMGN1, a nucleosome binding protein that destabilizes the higher-order chromatin structure, modulates the repair rate of ultraviolet light (UV)-induced DNA lesions in chromatin. Hmgn1(-/-) mouse embryonic fibroblasts (MEFs) are hypersensitive to UV, and the removal rate of photoproducts from the chromatin of Hmgn1(-/-) MEFs is decreased as compared with the chromatin of Hmgn1(+/+) MEFs; yet, host cell reactivation assays and DNA array analysis indicate that the nucleotide excision repair (NER) pathway in the Hmgn1(-/-) MEFs remains intact. The UV hypersensitivity of Hmgn1(-/-) MEFs could be rescued by transfection with plasmids expressing wild-type HMGN1 protein, but not with plasmids expressing HMGN1 mutants that do not bind to nucleosomes or do not unfold chromatin. Transcriptionally active genes, the main target of the NER pathways in mice, contain HMGN1 protein, and loss of HMGN1 protein reduces the accessibility of transcribed genes to nucleases. By reducing the compaction of the higher-order chromatin structure, HMGN1 facilitates access to UV-damaged DNA sites and enhances the rate of DNA repair in chromatin.