Warming weather changes the chemical composition of oat hulls.

The current threats of climate change are driving attention away from the petrochemical industry towards more sustainable and bio-based production processes for fuels and speciality chemicals. These processes require suitable low-cost starting material. One potential material assessed here is the oat hull. Its overall chemical composition has so far not been fully characterized. Furthermore, it is not known how it is affected by extreme weather events. Oat hulls (Kerstin and Galant varieties) grown during 'normal' weather years (2016 and 2017) are compared to the harvest of the warmer and drier year (2018). Standard methods for determination of plant chemical composition, with focus on carbohydrate composition, are utilized. Oat hulls grown in 'normal' weather conditions (2017) are rich in lignocellulose (84%), consisting of 35% hemicellulose, 25% lignin and 23% cellulose. Arabinoxylan was found to be the major biopolymer (32%). However, this composition is greatly influenced by weather variations during the oat growth phase. A lignocellulose reduction of 25% was recorded in the warmer and drier 2018 harvest. Additionally, a 6.6-fold increase in starch content, a four-fold increase in protein content and a 60% decrease in phenolic content was noted. Due to its high lignocellulose composition, with an exceptionally large hemicellulose fraction, the chemical composition of oat hulls is unique among agricultural by-products. However, this characteristic is significantly reduced when grown in warmer and drier weather, which could compromise its suitability for use in a successful biorefinery.

Activation of enzymes in organic media at low water activity by polyols and saccharides.

Horse liver alcohol dehydrogenase and alpha-chymotrypsin were deposited on a porous support material, Celite. After equilibration at a well-defined water activity, the catalytic activity was measured with diisopropyl ether as reaction medium. The effects of the presence of polyols and simple saccharides in the preparations were investigated. The additives caused a considerable increase in the amount of water bound to the preparation at a fixed water activity. At low water activities the catalytic activity was increased and at high water activity it was decreased by the additives. The presence of additives increased the ratio of alcoholysis-to-hydrolysis activity of chymotrypsin.

Substrate specificity of alpha-chymotrypsin-catalyzed esterification in organic media.

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.

Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine.

The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent Km values are the same for PC and LPC, namely 4.9 mM. Vmax is 82.5 and 10.4 nmol h(-1) mg(-1) for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.

Adsorption of lipase on polypropylene powder.

Adsorption of different lipases by EP-100 polypropylene powder from crude and pure lipase preparations was studied. Langmuir isotherms described the adsorption equilibria well both for protein and lipase activity adsorption. Adsorption isotherms for five different proteins all gave a similar saturation level of 220 mg protein per g carrier. Twelve commercial lipase preparations were tested for selectivity in the adsorption of lipase. For all preparations the selectivity factor was larger than one. In a crude lipase preparation from Pseudomonas fluorescence, the specific activity in solution decreased by two orders of magnitude after adsorption. The adsorption was not significantly influenced by pH changes in the adsorption buffer, indicating that hydrophobic and not electrostatic interactions are the dominating adsorption forces. Adsorption of a crude lipase from Candida rugosa (Sigma) was fast and equilibrium was reached in 30 and 100 min for protein and lipase activity adsorption respectively. Desorption in aqueous solution was negligible. Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent.

Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones.

The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.

Nucleophile specificity in alpha-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions.

Nucleophilic properties of amino-acid amides were studied systematically in acyl-transfer reactions catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-L-Ala-L-Ala-L-PheOMe as the acyl-group donor. In alpha-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: D-AlaNH2 < GlyNH2 < L-AlaNH2 < L-SerNH2 < L-ThrNH2 < L-HisNH2 < L-ValNH2 < L-LeuNH2 < L-TrpNH2 < L-MetNH2 < L-NvaNH2 < L-PheNH2 < L-IleNH2 < L-TyrNH2 < L-ArgNH2. In reactions catalyzed by subtilisin 72, the reactivities increase as follows: L-LeuNH2 < L-IleNH2 < L-ThrNH2 < L-ArgNH2 < L-TrpNH2 < L-NvaNH2 < L-ValNH2 < L-MetNH2 < L-AlaNH2 < L-SerNH2 < D-AlaNH2 < GlyNH2. In alpha-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of D-AlaNH2, L-ArgNH2 and L-TyrNH2. In reactions catalyzed by subtilisin 72, amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S1' subsite of subtilisin 72 and high specificity of this subsite in alpha-chymotrypsin.

Calorimetric studies on solid alpha-chymotrypsin preparations in air and in organic solvents.

Differential scanning calorimetry was the method to investigate the thermostability of chymotrypsin. The transition temperature decreased by approx. 30 degrees C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbitol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the enzyme was equilibrated through the apolar solvent, isooctane, stabilization of chymotrypsin was observed at high water activities, compared to equilibration in air. The presence of isooctane resulted in a remarkable stabilization of the chymotrypsin-sorbitol complex. A sorbitol concentration of 5 mmol/g of protein was prerequisite to induce stabilization when equilibrated through isooctane at high water activities. The transition enthalpy increased with increasing amounts of sorbitol. Different hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffer salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water activity 0.97. Variations on the hydration of the preparations both offered the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalyst whose hydration was suppressed due to its environment exhibited low enzymatic activity.

The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can be improved by optimisation of the temperature and by using a high co-substrate concentration.

The stereoselective reduction of ketones by alcohol dehydrogenase from Thermoanaerobium brockii was studied in organic reaction media. 2-Propanol was used as co-substrate to regenerate the coenzyme NADPH. The enantiomeric excess of the alcohol formed from the ketone decreased during the course of the reaction (from 53 to 0% e.e. in the formation of (R)-2-butanol). This was interpreted as being due to the reversibility of all the reactions involved. By using a large excess of 2-propanol this effect was suppressed. In the reduction of 2-butanone to (R)-2-butanol, the enantiomeric excess increased with increasing temperature, but in the reduction of 2-pentanone to (S)-2-pentanol the enantiomeric excess decreased with increasing temperature. The data were evaluated in terms of free energy of activation of the reaction pathways leading to the different possible products.

Fatty acid selectivity of a lipase purified from Vernonia galamensis seed.

Vernonia galamensis is an annual herb whose seed oil contains high levels of an epoxy fatty acid, vernolic (cis-12,13-epoxy cis-9-octadecenoic) acid. The seed also contains lipase activity in the dormant state. A lipase was purified from the seed and its substrate specificity studied in isooctane. The lipase shows pronounced selectivity for the native triacylglycerol, trivernolin. The rate of hydrolysis of triolein, the corresponding non epoxy triacylglycerol, is only 3% of that of trivernolin. In the acidolysis of tricaprylin using a mixture of fatty acids, the Vernonia lipase also showed selectivity for vernolic acid. Michaelis-Menten kinetics of the hydrolysis of triacylglycerols revealed that the observed high selectivity of the Vernonia lipase for trivernolin was mainly due to a higher Vmax for trivernolin. The Vmax value for the hydrolysis of trivernolin was 5 times higher than that for triolein. This novel substrate specificity is an adaptation by the seed lipase to the triacylglycerols of the seed oil that contain up to 80% vernolic acid.

Enzymatic synthesis of X-Phe-Leu-NH2 in low water content systems: influence of the N-alpha protecting group and the reaction medium composition.

The influence of eight different N-terminal protecting groups (For, Ac, Boc, Fmoc, Mal, Pheac, Aloc and Z) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH2 in organic media was studied. Groups such as Ac, For, Boc, Z, Mal, Pheac and Alloc always rendered good peptide yields (92% to 99%) either in acetonitrile or in ethyl acetate. Good correlations were found between molecular and physico-chemical characteristics of the N-alpha moiety such as the hydrophobicity (log P), ovality and dipole moment and the global reaction rate parameter k'. High k' values were obtained with the less hydrophobic groups, Ac, For and Mal, that have ovality values close to one and the highest dipole moments. Furthermore, it was found that the relative rate of hydrolysis and aminolysis of the acyl-enzyme intermediate expressed as the partition parameter p is affected by the N-alpha moiety of the acyl donor. Correlations between this parameter and the dipole moment of the protecting group were observed.

Tailoring the microenvironment of enzymes in water-poor systems.

Biocatalytic systems using enzymes in organic solvents open up the possibility of performing a whole range of reactions which would not normally occur under physiological conditions. The ability to perform reverse hydrolysis, or to convert substances relatively insoluble in aqueous environments on a scale of practical value in commercial applications are among those reactions for which water-poor systems are appropriate.

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

Organic solvent changes the chymotrypsin specificity with respect to nucleophiles.

In alpha-chymotrypsin-catalyzed acyl-transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of alpha-chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium.

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).

Two-enzyme system for the synthesis for 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate.

A combination of two enzymes, phospholipase D (PL D) and C (PL C), was investigated for the production of two lysophospholipids, 1-lauroyl-rac-glycerophosphate (1-LGP) and 1-lauroyl-dihydroxyacetonephosphate (1-LDHAP). The high transphosphatidylation ability of phospholipase D from Streptomyces sp. allowed the formation of 1-lauroyl-phosphatidylglycerol (1-LPG) and 1-lauroyl-phosphatidyldihydroxyacetone (1-LPDHA) from phosphatidylcholine (PC) and 1-monolauroyl-rac-glycerol (1-MLG) and 1-lauroyl-dihydroxyacetone (1-MDHA), respectively. A two-phase system, diethyl ether/water, was chosen for the convenience of the recovery of the water insoluble products. A similar two-phase system was used for hydrolysis of the complex phospholipids by phospholipase C form Bacillus cereus, which released both lysophospholipids. Only trace amounts of phosphatidic acid (PA) were detected showing that the enzyme is highly selective for the release of the diacylglycerol and 1-lauroyl-rac-glycerophosphate and 1-lauroyl-dihydroxyacetonephosphate.

Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine.

The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar V(max) values, suggesting that the formation of the phosphatidyl-enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca(2+), and the optimum concentration was approximately 40 mM CaCl(2). K(Ca)(app) was calculated to be 8.6+/-0.74 mM for the hydrolytic and 10+/-0.97 mM for the transphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca(2+) concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388+/-37 mM. Km(app) for the lysophosphatidylcholine depended on Ca(2+) concentration and fell between 1 and 3 mM at CaCl(2) concentrations from 4 to 40 mM. SDS, TX-100, and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentrations at or above that of lysophosphatidylcholine.

Enzymatic fatty acid exchange in digalactosyldiacylglycerol.

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).

Side reactions in enzymatic peptide synthesis in organic media: effects of enzyme, solvent, and substrate concentrations.

The progress of enzymatic peptide synthesis catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the L-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH2 or ZAlaAlaPheXaaNH2 occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'1-specificity of alpha-chymotrypsin and the S1-specificity of subtilisin 72, while the S1-specificity of alpha-chymotrypsin and the S'1-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the alpha-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the alpha-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed at the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.