Enhanced Enzyme Activity and Enantioselectivity of Lipases in Organic Solvents by Crown Ethers and Cyclodextrins

Mine, Yurie; Fukunaga, Kimitoshi; Itoh, Kyoko; Yoshimoto, Makoto; Nakao, Katsumi; Sugimura, Yoshiaki

doi:10.1263/jbb.95.441

Cited by 10 publications

(13 citation statements)

References 32 publications

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“…1). This finding is in agreement with those reported by Mine et al (2003), who did not observe variation of the E value for lipase BC, lyophilized with 18-crown-6 in the transesterification between 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate. Contrary to lipase BC, CALB co-lyophilized with the crown ether shows an increase of its E value (inset Fig.…”

Section: Results and Discussion Transesterification Activity And Enansupporting

confidence: 94%

“…In particular, the transesterification activity in toluene increased up to 2.5-and 1.4-fold for lipases BC and CALB, respectively. This finding agrees with those reported by other research groups with other hydrolases (Mine et al, 2003;Santos et al, 2001;van Unen et al, 2002). Figures 1 and 2 show that the activity increase was obtained using 18-crown-6/lipase molar ratios 100.…”

Section: Results and Discussion Transesterification Activity And Enansupporting

confidence: 92%

“…A method, commonly employed to overcome this problem and to preserve the enzymatic activity, is the use of proper additives in the lyophilization buffer. Crown ethers (especially 18-crown-6) are among the additives that have a beneficial effect on the activity and enantioselectivity (Mine et al, 2003;Persson et al, 2002;Santos et al, 2001;Tsukube et al, 2001;van Unen et al, 1998) of hydrolases in organic solvents. Concerning the way by which they improve the enzyme activity, van Unen et al (2002) have suggested two simultaneous mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Secundo

Barletta

Dumitriu

et al. 2006

Biotech & Bioengineering

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Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.

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Section: Results and Discussion Transesterification Activity And Enansupporting

confidence: 94%

Section: Results and Discussion Transesterification Activity And Enansupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Secundo

Barletta

Dumitriu

et al. 2006

Biotech & Bioengineering

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“…Much effort has been focused on strategies to overcome this issue, including enzyme immobilization on porous and non-porous solid supports 12,13 , chemical modification of the enzymes' surfaces to improve compatibility with solvents 14 , protein engineering 2 , and enzyme co-lyophilization with different types of excipients, such as cyclodextrins 10,15 , crown ethers [15][16][17] , and inorganic salts [18][19][20] . Specifically, the co-lyophilization of enzymes with inorganic salts from aqueous solution, termed salt activation, has been remarkably successful.…”

Section: Introductionmentioning

confidence: 99%

Immobilization of Candida antarctica Lipase B on fumed silica

Cruz

Pfromm

Rezac

2009

Process Biochemistry

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Enzymes are usually immobilized on solid supports or solubilized when they are to be used in organic solvents with poor enzyme solubility. We have reported previously on a novel immobilization method for s. Carlsberg on fumed silica with results that reached some of the best previously reported catalytic activities in hexane for this enzyme. Here we extend our method to Candida antarctica lipase B (CALB) as an attractive target due to the many potential applications of this enzyme in solvents. Our CALB/fumed silica preparations approached the catalytic activity of commercial Novozym 435 for a model esterification in hexane at 90wt% fumed silica (relative to the mass of the preparation). An intriguing observation was that the catalytic activity at first increases as more fumed silica was made available to the enzyme but then decreased precipitously when 90wt% fumed silica was exceeded. This was not the case for s. Carlsberg where the catalytic activity leveled off at high relative amounts of fumed silica. We determined adsorption kinetics, performed variations of the pre-immobilization aqueous pH, determined the stability, and applied fluorescence microscopy to the preparations. A comparison with recent concepts by Gross et al. may point towards a rationale for an optimum intermediate surface coverage for some enzymes on solid supports.

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“…The preferred matrices for immobilization include macroporous polypropylene particles (Bosley and Peilow, 1997), hydrophilic silicon wafers (van der Veen et al, 2007), microemulsions and organogels (Zoumpanioti et al, 2008). Additional efforts include improving compatibility with the solvents by chemical modification of the enzymes' surface (Sheldon et al, 2005), protein engineering (Hudson et al, 2005), and co-lyophilization of the enzyme with various adjuvants, such as cyclodextrin (Ghanem, 2003;Mine et al, 2003), inorganic salts (Lindsay et al, 2002(Lindsay et al, , 2004, and crown ethers (Mine et al, 2003;Santos et al, 2001;Secundo et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Enantioselective transesterification by Candida antarctica Lipase B immobilized on fumed silica

Kramer

Cruz

Pfromm

et al. 2010

Journal of Biotechnology

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Enzymatic catalysis to produce molecules such as perfumes, flavors, and fragrances has the advantage of allowing the products to be labeled "natural" for marketing in the U.S., in addition to the exquisite selectivity and stereoselectivity of enzymes that can be an advantage over chemical catalysis. Enzymatic catalysis in organic solvents is attractive if solubility issues of reactants or products, or thermodynamic issues (water as a product in esterification) complicate or prevent aqueous enzymatic catalysis. Immobilization of the enzyme on a solid support can address the generally poor solubility of enzymes in most solvents.We have recently reported on a novel immobilization method for Candida antarctica Lipase B on fumed silica to improve the enzymatic activity in hexane. This research is extended here to study the enantioselective transesterification of (RS)-1-phenylethanol with vinyl acetate. The maximum catalytic activity for this preparation exceeded the activity (on an equal enzyme amount basis) of the commercial Novozyme 435® significantly. The steady-state conversion for (R)-1-phenylethanol was about 75% as confirmed via forward and reverse reaction. The catalytic activity steeply increases with increasing nominal surface coverage of the support until a maximum is reached at a nominal surface coverage of 230%. We hypothesize that the physical state of the enzyme molecules at a low surface coverage is dominated in this case by detrimental strong enzyme-substrate interactions. Enzyme-enzyme interactions may stabilize the active form of the enzyme as surface coverage increases while diffusion limitations reduce the apparent catalytic performance again at multi-layer coverage. The temperature-, solvent-, and long-term stability for CALB/fumed silica preparations showed that these preparations can tolerate temperatures up to 70°C, continuous exposure to solvents, and long term storage.2

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Enhanced Enzyme Activity and Enantioselectivity of Lipases in Organic Solvents by Crown Ethers and Cyclodextrins

Cited by 10 publications

References 32 publications

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Immobilization of Candida antarctica Lipase B on fumed silica

Enantioselective transesterification by Candida antarctica Lipase B immobilized on fumed silica

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