Cellulose recalcitrance toward saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural cellulose-III allomorph that is less recalcitrant toward enzymatic hydrolysis. Here, we characterize the hydrolytic activity of a thermophilic cellulolytic microbe, Thermobifida f usca, derived cellulase on cellulose-III. Up to 2-fold improved activity was observed for homologously expressed T. f usca cellulase enzymes on cellulose-III. Surprisingly, T. f usca exocellulases like Cel6B alone had lower activity on cellulose-III. We hypothesized that increased activity of T. f usca cellulases on cellulose-III arises mostly due to enhanced endocellulase activity and improved synergism between endo/exocellulases. Representative T. fusca endocellulase (Cel5A) and exocellulase (Cel6B) were heterologously expressed in Escherichia coli, purified, and systematically characterized for synergistic activity on cellulose-III. Hydrolytic activity assays confirmed increased activity of Cel5A on cellulose-III and improved endo/exo synergistic activity for various combinations of Cel6B/Cel5A. We finally conducted a two-step restart hydrolysis assay to also confirm if increased endoactivity results in a endotreated cellulose-III that is amenable toward increased Cel6B activity. This work provides a mechanistic basis for increased synergistic cellulase activity on cellulose-III and provides a rationale for focusing future T. f usca enzyme engineering efforts toward potentially rate-limiting exocellulases like Cel6B.
Cannabidiol (CBD) hemp seed oil is a commercial raw material with antioxidant and anti-inflammatory benefits that has been formulated into body wash and skin care products. The biggest analytical challenge is how to simultaneously quantify CBD and hemp seed oil as they deposited on the skin surface. CBD is easily separated and quantified from skin surface extracts via a HPLC-mass spectrometry methodology. However, the structural skeleton of triacylglycerides (TAGs) in hemp seed oil is same as those from the skin surface sebum. The strong hydrophobicity with subtle structural difference challenges their separation. In this project, a new reverse phase HPLC-high resolution mass spectrometry methodology was developed with a strong mobile phase normal propanol. The separated hemp seed oil TAGs in the chromatogram were identified and characterized using data-dependent acquisition (DDA) technology. Based on the daughter ion characterization, the separated peak with an ammonium adduct at 890.7226 [M + NH4]+ was confirmed as the parent ion of glycerol with three omega-3 fatty acid chains. This is the first time TAG structure with direct HPLC-tandem mass spectrometry technology has been elucidated without a hydrolysis reaction. The confirmed TAG structure with an ammonium adduct at 890.7226 ± 0.0020 can be used as a representative chemical marker for the hemp seed oil quantification.
Magnesium silicate is an inorganic compound used as an ingredient in product formulations for many different purposes. Since its compatibility with other components is critical for product quality and stability, it is essential to characterize the integrity of magnesium silicate in different solutions used for formulations. In this paper, we have determined the magnitude of dissociation of synthetic magnesium silicate in solution with positively charged, neutral, and negatively charged compounds using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). The EDS results were verified through Monte Carlo simulations of electron-sample interactions. The compounds chosen for this study were positively charged cetylpyridinium chloride (CPC), neutral lauryl glucoside, and negatively charged sodium cocoyl glutamate and sodium cocoyl glycinate since these are common compounds used in personal care and oral care formulations. Negatively charged compounds significantly impacted magnesium silicate dissociation, resulting in physio-chemical separation between magnesium and silicate ions. In contrast, the positively charged compound had a minor effect on dissociation due to ion competition, and the neutral compound did not have such an impact on magnesium silicate dissociation. Further, when the magnesium ions are dissociated from the synthetic magnesium silicate, the morphology is changed accordingly, and the structural integrity of the synthetic magnesium silicate is damaged. The results provide scientific confidence and guidance for product development using synthetic magnesium silicate.
Flaxseed oil is a popular natural ingredient in cosmetics due to its proven nutritional and skin health benefits. It has been shown to exhibit antioxidant and anti-inflammatory activities when deposited effectively on the skin surface. However, the complexity of triacylglyceride structures in flaxseed oil has made it difficult to fully characterize them. To address this issue, a new method was developed that combined gas and liquid chromatography with mass spectrometry to identify a chemical marker of flaxseed oil that can be used to quantify its deposition on skin. This method was applied to study the deposition differences on pig and human skins. The use of flaxseed oil in skincare products, in combination with a cationic polymer, has been demonstrated to be effective in improving skin health.
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