Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Bioethanol is an appealing alternative to petroleum-based liquid fuel due to drivers such as environmental regulations and government mandates. Second generation lignocellulosic feedstocks are abundant, but their resistance to hydrolysis continues to be problematic. Different pretreatments have been proposed to increase cellulose reactivity. Softwood pine autohydrolyzed at different severities was subjected to further treatment to increase fibre reactivity. Liquid hot water is most effective at removing barriers, with the highest increase in sugar yield after enzymatic hydrolysis. Alkaline (NaOH) is found to be the worst option compared to dilute acid and organosolv. In addition, higher chemical concentrations and longer treatment times do not guarantee higher enzymatic hydrolysis yield. Process modifications such as fiber washing and multistage enzymatic hydrolysis are observed to be effective at increasing yield. However, more research is required to bring the enzymatic hydrolysis yield to a level where commercialization is feasible.

Bioethanol is an appealing alternative to petroleum-based liquid fuel due to drivers such as environmental regulations and government mandates. Second generation lignocellulosic feedstocks are abundant, but their resistance to hydrolysis continues to be problematic. Different pretreatments have been proposed to increase cellulose reactivity. Softwood pine autohydrolyzed at different severities was subjected to further treatment to increase fibre reactivity. Liquid hot water is most effective at removing barriers, with the highest increase in sugar yield after enzymatic hydrolysis. Alkaline (NaOH) is found to be the worst option compared to dilute acid and organosolv. In addition, higher chemical concentrations and longer treatment times do not guarantee higher enzymatic hydrolysis yield. Process modifications such as fiber washing and multistage enzymatic hydrolysis are observed to be effective at increasing yield. However, more research is required to bring the enzymatic hydrolysis yield to a level where commercialization is feasible.

Construction of nanoassemblies from degradable components is desired for packaging and controlled release of active therapeutics, and eventual biodegradability in vivo. In this study, shell crosslinked micelles composed of biodegradable poly(lactide) (PLA) core were prepared by the self-assembly of an amphiphilic diblock copolymer synthesized by a combination of ring opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Enzymatic degradation of the PLA cores of the nanoparticles was achieved upon the addition of proteinase K (PK). Kinetic analyses and comparison of the properties of the nanomaterials as a function of degradation extent will be discussed. Building upon our findings from selective-excavation of the PLA core, enzyme- and redox-responsive nanoparticles were constructed for the encapsulation and stimuli-responsive release of an antitumor drug. This potent chemotherapeutic, otherwise poorly soluble in water was dispersed into aqueous solution by the supramolecular co-assembly with an amphiphilic block copolymer, and the release from within the core of these nanoparticles were gated by crosslinking the hydrophilic shell region with a reduction-responsive crosslinker. Enzyme- and reduction-triggered release behavior of the antitumor drug was demonstrated along with their remarkably high in vitro efficacy. As cationic nanoparticles are a promising class of transfection agents for nucleic acid delivery, in the next part of the study, synthetic methodologies were developed for the conversion of the negatively-charged shell of the enzymatically-degradable shell crosslinked micelles to positively-charged cationic nanoparticles for the complexation of nucleic acids. These degradable cationic nanoparticles were found to efficiently deliver and transfect plasmid DNA in vitro. The hydrolysis of the PLA core and crosslinkers of the nanocarriers may provide a mechanism for their programmed disassembly within endosomes, which would in-turn promote endosomal disruption by osmotic swelling, and release of active therapeutics from the polymeric assemblies. In the last part, a comparative degradation study was performed between the anionic and cationic micellar assemblies in the presence of two model enzymes, and electrostatic interaction-mediated preferential hydrolysis was demonstrated between the oppositely-charged enzyme-micelle pairs. These findings may be of potential significance toward the design of charge-mediated enzyme-responsive nanomaterials that are capable of undergoing environmentally-triggered therapeutic release, disassembly or morphological alterations under selective enzyme conditions.

Racemic metyhl 3-cyclohexene-1-carboxylate was resolved via enzymatic hydrolysis to afford the enantiomerically enriched 3-cyclohexene-1-carboxylic acid with PLE (S-configuration), HLE (S-configuration), CCL (S-configuration) and PPL (R-configuration) . The nucleoside&amp / #65533 / s precursor, 5-(hydroxymethyl)-2-cyclohexen-1-ol (19), was synthesized by iodolactonization, followed by iodine elimination and the reduction of the lactone. In connection with this work, alpha,beta-unsaturated and saturated cyclic ketones were selectively oxidized on alpha&#039 / - and alpha-positions using Mn(OAc)3 and Pb(OAc)4, respectively. The resultant racemic alpha&#039 / - and alpha-acetoxylated substrates were resolved into corresponding enantiomerically enriched alpha&#039 / - and alpha-hydroxylated and acetoxylated compounds via PLE hydrolysis.

&amp / #945 / ,&amp / #946 / -Unsaturated cyclic ketones were selectively oxidized on &amp / #945 / &#039 / - positions using Mn(OAc)3 and Pb(OAc)4, respectively. The resultant racemic &amp / #945 / &#039 / -acetoxylated substrates were resolved into corresponding enantiomerically enriched &amp / #945 / &#039 / -hydroxylated and &amp / #945 / &#039 / -acetoxylated compounds via PLE hydrolysis. &amp / #945 / &#039 / -Hydroxylated compounds are racemized quickly, so they were acetylated with acetyl chloride and pyridine in situ to give the corresponding &amp / #945 / &#039 / -acetoxylated compounds. Resultant &amp / #945 / &#039 / -acetoxy &amp / #945 / ,&amp / #946 / -unsaturated cyclic ketones reacted with excess amount of diazomethane under the catalsts of Pd(OAc)2 to give the resulting bicyclic diastereomeric products. At the end of the experiment, Enantiomeically enriched 2-oxobicyclo[3.1.0]hexan-3-yl acetate and 2-oxobicyclo[4.1.0]heptan-3-yl acetate were chemoenzymatically synthesized.

Sodium hydroxide can cause cellulose to swell and can even dissolve cellulose in a narrow range of the phase diagram. It was found that for cellulose with low to moderate degree of polymerization, the maximal solubility occurs with 8~10% soda solution. In recent years, researchers found that sodium hydroxide with urea at cold temperature can dissolve cellulose better than sodium hydroxide alone. However, the lack of sufficient understanding of the NaOH and NaOH/urea dissolution process significantly constrains its applications. In order to fully understand the cellulose dissolution in alkali system, there are several aspects of problems that need to be addressed. Our focus in this study is in the interaction of cellulose with alkali solution at low temperatures, the improvement of its solubility, and the effect of hemicellulose and lignin.

Low cost cellulosic wastes like paper sludge, municipal wastes, solid wastes from food, packing etc. contain a high amount of cellulose which can be converted to bioethanol by two steps: (1) solubilization of cellulosic fibers to monosaccharides (2) conversion of monosachharides to bioethanol via fermentation. At present the implementation of this technology has been deterred by high cost for enzymes. Enzymatic hydrolysis of cellulosic fibers shows a biphasic behavior with an initial fast step followed by a slow step leading to low cellulose conversion rates. Low hydrolytic conversion rates necessitate the use of a high enzyme dosage to obtain meaningful cellulose conversion rates which make the implementation of this entire technology economically infeasible. The objective of this study is to get a better understanding of the mechanism of enzymatic hydrolysis of fibers to glucose and to investigate the effect of cationic polymers on enzymatic hydrolysis rates. To achieve the first objective, we performed experiments so as to study changes in morphological and physiochemical properties like fiber length, percentage of fines, crystallinity index, kink angle, kink index, mean curl, total organic carbon and glucose production with time. We used bleached kraft softwood, hardwood, and unbleached softwood fiber as cellulosic substrate and pergalase as cellulase enzyme. All of the experiments were carried out at experimental conditions of a temperature of 50 .C and a pH of 5.0 which maximize enzymatic activity. We studied the impact of recycling and refining on hydrolysis rates by measuring total organic carbon and glucose production. We found that refining increases enzymatic conversion rates by about as much as 20 %, however refining being energy intensive makes its implementation economically unfavorable. We found a novel way of enhancing hydrolysis rates by the use of cationic polyacrylamides. The effect of cationic polacrylamides was studied on both hardwood and softwood fibers at similar experimental conditions. Cationic polyacrylamides produced a maximum rate increase of 20 % in hydrolytic conversion rates for hardwood fibers. Even though, the increase in hydrolysis rates for softwood fibers was smaller than hardwood fibers, it was still significant. We further studied the effect of parameters like polymer concentration, cationicity and molecular weight to find a relation between properties of polymers and the increase in enzymatic hydrolysis.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2010. / Härtill 3 uppsatser.

Many aspects associated with conversion of lignocellulose to biofuels and other valuable products have been investigated to develop the most effective processes for biorefineries. The goal of this research was to improve the efficiency and effectiveness of the lignocellulose conversion process by achieving a more basic understanding of pretreatment and enzymatic hydrolysis at high solids, including kinetic modeling and separation and recovery of glucose. Effects of NaOH pretreatment conditions on saccharide yields from enzymatic hydrolysis were characterized in low- and high-solids systems. Factors associated with pretreatment and hydrolysis were investigated, including duration of pretreatment at different temperatures and NaOH loadings, as well as different solids and enzyme loadings. Under relatively mild pretreatment conditions, corn stover composition was essentially equivalent for all time and temperature combinations; however, components were likely affected by pretreatment, as differences in subsequent cellulose conversions were observed. Flushing the hydrolyzate and reusing the substrate was also studied as a method for inhibitor mitigation while increasing overall glucose yields. Flushing the PCS throughout the hydrolysis reaction eliminated the need to wash the pretreated biomass prior to enzymatic hydrolysis when supplementing with low doses of enzyme, thus reducing the amount of process water required. The robustness of an established kinetic model was examined for heterogeneous hydrolysis reactions in high-solids systems. Michaelis-Menten kinetics is the traditional approach to modeling enzymatic hydrolysis; however, high-solids reactions violate the main underlying assumption of the equation: that the reaction is homogeneous in nature. The ability to accurately predict product yields from enzymatic hydrolysis in high-solids systems will aid in optimizing the conversion process. Molecularly-imprinted materials were studied for use in both bulk adsorption and in column chromatography separations. Glucose-imprinted materials selectively adsorbed glucose compared xylose by nearly 4:1. Non-imprinted materials were neither selective in the type of sugar adsorbed, nor were they capable of adsorbing sugar at as high a capacity as the glucose-imprinted materials. Liquid chromatography with imprinted materials was not a suitable means for separating glucose from solution under the conditions investigated; however, many factors impact the effectiveness of such a separation process and warrant further investigation.