- Ionic-surfactant-coated subtilisin: Activity, enantioselectivity, and application to dynamic kinetic resolution of secondary alcohols
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In this work, we explored the utility of ionic-surfactant-coated Bacillus licheniformis subtilisin (ISCBLS) as the catalyst for the dynamic kinetic resolution of secondary alcohols. ISCBLS was prepared by freeze-drying Bacillus licheniformis subtilisin wi
- Kim, Kyungwoo,Lee, Eungyeong,Kim, Cheolwoo,Park, Jaiwook,Kim, Mahn-Joo
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p. 8836 - 8844
(2017/11/03)
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- NON-AQUEOUS ENZYME-POLYMER CONJUGATE SOLUTIONS AND RELATED METHODS
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An enzyme-polymer conjugate shows increased activity and molecular dissolution in non-aqueous solvents enabling enzyme mediated catalysis in non-aqueous solutions. The inventions described in this specification relate to enzyme-polymer conjugates, organic
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Paragraph 0111-0113
(2018/01/11)
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- Urea treated subtilisin as a biocatalyst for transformations in organic solvents
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Abstract Subtilisin lyophilized from its solution in aqueous buffer in the presence of 6 M urea showed up to 50-fold increase (as compared to lyophilized subtilisin not subjected to urea treatment) in its initial rate of a transesterification reaction in anhydrous n-hexane. The lyophilization time controlling the extent of 'drying' was an important parameter. The urea treated subtilisin had five times shorter half life during heating at 100 C in hexane. The change in conformation was also reflected in its 92-fold higher activity at 15 C as compared to merely 28-fold higher activity at 45 C. The comparative enantioselectivity of urea treated subtilisin during kinetic resolution of 1-phenylethanol was expectedly lower. Its enantioselectivity during kinetic resolution of a natural substrate N-acetyl-(R,S)-phenylalanine ethyl ester in hexane was higher. Urea treated subtilisin also showed higher catalytic promiscuity during an aldol condensation. CD studies in both far UV and near UV region were also carried out to compare the two structures.
- Mukherjee, Joyeeta,Mishra, Prasant,Gupta, Munishwar N.
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p. 1976 - 1981
(2015/03/30)
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- Enhancing the catalytic efficiency of subtilisin for transesterification by dual bioimprinting
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Bioimprinting is a technique in which an aqueous solution of a protein molecule along with the imprint molecule is dried to remove bulk water. Subtilisin was dissolved in an aqueous buffer with a substrate analog and precipitated with the substrate alcoho
- Mukherjee, Joyeeta,Gupta, Munishwar N.
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p. 4397 - 4401
(2015/06/22)
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- Protease activation in glycerol-based deep eutectic solvents
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Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmol min -1 g-1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-l-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally.
- Zhao, Hua,Baker, Gary A.,Holmes, Shaletha
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experimental part
p. 163 - 167
(2012/07/01)
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- Soluble, folded and active subtilisin in a protic ionic liquid
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The activity of proteases chymotrypsin and subtilisin dissolved in a range of protic hydroxylalkylammonium ionic liquids was tested against the model substrate APEE (N-acetyl-l-phenylalanine ethyl ester); activity was only observed for subtilisin in diethanolammonium chloride (DEA Cl), while chymotrypsin was not active in any PIL tested.
- Falcioni, Francesco,Housden, Hazel R.,Ling, Zhenlian,Shimizu, Seishi,Walker, Adam J.,Bruce, Neil C.
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supporting information; experimental part
p. 749 - 751
(2010/06/12)
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- Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents
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The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number Acatsup
- Hudson, Elton P.,Eppler, Ross K.,Beaudoin, Julianne M.,Dordick, Jonathan S.,Reimer, Jeffrey A.,Clark, Douglas S.
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experimental part
p. 4294 - 4300
(2009/09/30)
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- Enhanced activity of α-chymotrypsin in organic media using designed molecular staples
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We report the enhancement of α-chymotrypsin activity in organic solvents using modified peptides bearing two crown ethers. The transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol was used as model reaction. Co-lyophilization of cro
- Tremblay, Mélanie,C?té, Simon,Voyer, Normand
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p. 6824 - 6828
(2007/10/03)
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- Nanophase separated amphiphilic microbeads
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The synthesis of nonporous nanophase separated amphiphilic microbeads was investigated. The synthesis was performed with a mixture of α,ω- methacrylate-terminated poly-dimethylsiloxane (MA-PDMS-MA), trimethylsilylated 2-hydroxyethylacrylate (TMSOEA) and t
- Savin, Gabriela,Bruns, Nico,Thomann, Yi,Tiller, Joerg C.
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p. 7536 - 7539
(2008/02/01)
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- Fluorometric Assay Protocol for Protease-Catalyzed Transesterification Reactions in Organic Solvents
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A flourometric assay protocol for a subtilisin-catalyzed transesterification reaction in n-hexane has been developed. The method makes use of a Michael acceptor that forms a fluorescent adduct with thiophenol, one of the products generated in the transest
- Han, Min Su,Jung, Sang Oh,Kim, Mahn-Joo,Kim, Dong H.
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p. 2853 - 2855
(2007/10/03)
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- Enhancement of catalytic activity of chemically modified subtilisin Carlsberg in benzene by adjustment of lyophilization conditions
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Alteration of the lyophilization conditions significantly enhanced not only the activity of insoluble native subtilisin Carlsberg but also that of solubilized enzyme in benzene. The catalytic activity (k(cat)/K(m)) for the transesterification reaction of
- Kwon, Oh Hyeong,Imanishi, Yukio,Ito, Yoshihiro
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p. 1277 - 1282
(2007/10/03)
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- Structure and function of subtilisin BPN' solubilized in organic solvents
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Enzyme structure and function have been studied for subtilisin BPN' solubilized in organic solvents by ion pairing with low concentrations of an anionic surfactant (Aerosol OT) in the absence of reversed micelles. Soluble subtilisin shows strikingly different behavior in octane and tetrahydrofuran (THF). In octane, the k(cat)/K(m) for the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) is 370 M-1 s-1, within one order of magnitude of the enzyme's hydrolytic activity in water. Moreover, the observed half-life of the soluble enzyme in octane is nearly three orders of magnitude greater than in water, presumably because of the absence of autolysis in the organic solvent. In contrast, the catalytic efficiency of the enzyme dissolved in the polar solvent THF is 0.04 M-1 s-1, and the enzyme loses 99% of its activity within 10 min. Comparable enzyme inactivation could also be observed in octane, but only at elevated temperatures such as 70 °C. Therefore, the mechanisms of deactivation of the soluble enzyme were investigated in both octane and THF. Kinetic and spectroscopic (CD and EPR) studies support the existence of multiple inactive forms of the soluble enzyme in THF at 25 °C and in octane at 70 °C. Notably, in both cases a denatured form can be renatured in anhydrous octane at 25 °C, the first demonstration of enzyme renaturation in a bulk organic solvent. A model explaining the THF- and thermally-induced inactivation processes of soluble subtilisin BPN' is proposed, and the apparent reasons for the exceptionally high activity and stability of the soluble enzyme in octane are discussed.
- Wangikar, Pramod P.,Michels, Peter C.,Clark, Douglas S.,Dordick, Jonathan S.
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- Methanol dramatically enhances serine protease activity under anhydrous conditions
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The catalytic activity of rigorously dried lyophilised subtilisin Carlsberg and α-chymotrypsin in anhydrous solvents is enhanced dramatically on addition of dry methanol, while ethanol and propanol produce no effect.
- Hutcheon, Gillian A.,Parker, Marie Claire,James, Allan,Moore, Barry D.
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p. 931 - 932
(2007/10/03)
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- Dipole formation and solvent electrostriction in subtilisin catalysis
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The transition state for subtilisin-catalyzed transesterification was probed by high-pressure kinetic studies in solvents spanning a wide range of dielectric constants. The electrostatic model of Kirkwood described the solvent effects and gave a lower lim
- Michels, Peter C.,Dordick, Jonathan S.,Clark, Douglas S.
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p. 9331 - 9335
(2007/10/03)
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- Cross-linked crystals of subtilisin: Versatile catalyst for organic synthesis
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Cross-linked enzyme crystals (CLECs) of subtilisin exhibit excellent activity in aqueous and various organic solvents. This catalyst is more stable than the native enzyme in both aqueous and mixed aqueous/organic solutions. Subtilisin-CLEC was shown to be a versatile catalyst. It was used for the syntheses of peptides and peptidomimetics, mild hydrolysis of amino acid and peptide amides, enantio- and regioselective reactions, and transesterifications.
- Wang, Yi-Fong,Yakovlevsky, Kirill,Zhang, Bailing,Margolin, Alexey L.
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p. 3488 - 3495
(2007/10/03)
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- The mechanistic dissection of the plunge in enzymatic activity upon transition from water to anhydrous solvents
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Subtilisin Carlsberg dissolved in aqueous solution is several orders of magnitude more active than the enzyme suspended in anhydrous acetonitrile. In order to ascertain why, we employed crystalline subtilisin lightly cross-linked with glutaraldehyde as a catalyst in both aqueous and organic media. The structure of this crystalline enzyme in acetonitrile had been previously found to be virtually identical to that in water, thus ruling out solvent induced conformational changes as the cause of the enzymatic activity drop. Titration of the competent active centers of subtilisin revealed that k(cat)/K(M) is solely responsible for this activity plunge. Quantitative mechanistic analysis of the 7-order-of-magnitude difference in (cat)/K(M) values between subtilisin dissolved in water and cross-linked subtilisin crystals suspended in anhydrous acetonitrile allowed accounting for at least 5.6 orders of magnitude. This drastic decline is due to (i) a marked shift in the activity vs pH profile of the cross-linked crystalline enzyme compared to its soluble counterpart; (ii) different (far less favorable in acetonitrile than in water) energetics of substrate desolvation; and (iii) very low thermodynamic activity of water in anhydrous acetonitrile resulting in a much more rigid and thus less active enzyme.
- Schmitke, Jennifer L.,Wescott, Charles R.,Klibanov, Alexander M.
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p. 3360 - 3365
(2007/10/03)
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- Inversion of enantioselectivity of serine proteases
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The enantioselectivity of serine proteases in the transesterification of N-acetyl-(D,L)-phenylalanine esters with propan-1-ol in cyclohexane is strongly influenced by the leaving ability of the alcoholate group.Moreover the enantioselectivity is greatly influenced by the addition of small amounts (0.33 M) of organic additives.Addenda with a small molecular volume like e.g. ethanol and acetonitrile increase the rate for the L-enantiomer whereas alcohols with bulky alkyl groups like e.g. tert-butanol and 2-methylbutan-2-ol enhance the activity of the enzyme towards the D-enantiomer.This enables a rational tuning of the enantioselectivity as was demonstrated for four different proteases (α-chymotrypsin, subtilisin Carlsberg, Aspergillus oryzae protease, and elastase).
- Broos, Jaap,Engbersen, Johan F. J.,Verboom, Willem,Reinhoudt, David N.
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p. 255 - 258
(2007/10/02)
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- The Effect of Crown Ethers on Enzyme-catalysed Reactions in Organic Solvents
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Crown ethers considerably enhance the rate of the α-chymotrypsin-catalysed transesterification of N-acetyl-L-phenylalanine ethyl ester (N-Ac-L-Phe-OEt) with propan-1-ol in n-octane; with subtilisin the effect is somewhat less pronounced.
- Reinhoudt, David N.,Eendebak, Anke M.,Nijenhuis, Wilma F.,Verboom, Willem,Kloosterman, Marcel,Shoemaker, Hans E.
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p. 399 - 400
(2007/10/02)
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