PhD Pavlidis

.

, 2011

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() , , . , ...

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, . : , , , . , , . , , , , . . , , ... , . . , . , , . , ( ). , , . . 2004 : . , , , , , Ali Taha, . , . Uwe Bornscheuer Torge Vorhaben Greifswald , IKY DAAD .

iii

Enrico Maccallini, Calambria () FTIR. Petra Rudolf Groningen () XPS. . . . , . . . . , , , , . , . , , , , , , , .

iv

: 1. I.V. Pavlidis, T. Vorhaben, U.T. Bornscheuer, G. K. Papadopoulos, H. Stamatis. The influence of functionalized carbon based nanomaterials on catalytic behavior and structure of hydrolases. (2011) Submitted 2. I.V. Pavlidis, T. Vorhaben, T. Tsoufis, P. Rudolf, U.T. Bornscheuer, D. Gournis, H. Stamatis. Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials. (2011) Submitted 3. 4. 5. I.V. Pavlidis, K. Tzafestas, H. Stamatis. Water-in-ionic liquid microemulsion-based organogels as novel matrices for enzyme immobilization. Biotechnology Journal 5 (2010) 805-812. I.V. Pavlidis, T. Tsoufis, A. Enotiadis, D. Gournis, H. Stamatis. Functionalized multi-wall carbon nanotubes for lipase immobilization. Advanced Engineering Materials 10 (2010): B179-B183. .V. Pavlidis, D. Gournis, G.K. Papadopoulos, H. Stamatis. Lipases in water-in-ionic liquid microemulsions. Structural and activity studies. Journal of Molecular Catalysis B: Enzymatic 60 (2009): 50-56. : 1. 2. I.V. Pavlidis, T. Tsoufis, D. Gournis, H. Stamatis. Enzyme immobilization on chemically functionalized multi-walled carbon nanotubes. New Biotechnology 25 (2009): S131 M. Katsoura, M. Patila, I.V. Pavlidis, H. Stamatis. Structural and stability studies of lipases in ionic liquids. New biotechnology 25 (2009): S131 Wiley: I.V. Pavlidis, A.A. Tzialla, A. Enotiadis, H. Stamatis, D. Gournis. Chapter 2: Enzyme immobilization on layered and nanostructured materials (2010) Biocatalysis in polymer chemistry (K. Loos ed.), Wiley-VCH Verlag, pp.35-64 ISBN-10: 3-527-32618-9. 17 :

2 9 2 4

v

vi

. , . : . . ( [bmim]PF6) Tween 20. , . . -, - , . , . () (HPMC) . . , ( 200 ) ( 25 ). , . (CNTs) (.. , , ) .

vii

, 1.65 mg mg . . CNTs . , , , . 75 . . , . -. , , . ( , ) , . , . , . , . , .

viii

Abstract

AbstractNanostructured systems are of considerable research interest in the field of enzyme biotechnology. The aim of this thesis is to study and understand the structure-function relationship of hydrolytic enzymes in nanostructured systems, leading to the rational design of novel nanobiocatalytic systems. The study focuses on two types of nanostructured systems: water in ionic liquid microemulsions and carbon-based nanomaterials. Water in ionic liquid microemulsions are novel water-nanodispersion systems which combine the advantages of both microemulsions and ionic liquids (IL). Such microemulsions can be formed using the ionic liquid [bmim]PF6 and the non-ionic surfactant Tween 20. Lipases entrapped in these IL microemulsions exhibited improved catalytic behaviour compared to other microheterogeneous systems, indicating the positive effect of the organized nanostructures on the catalytic behavior of enzymes. The composition of IL microemulsions affects significantly the catalytic characteristics and the structure of encapsulated lipases. Lipases in IL microemulsions adopt a rigid structure rich in -sheets, which is responsible for the observed increase in thermostability. At the same time, lipases retain their -helices, which are associated with their increased catalytic activity. A significant advantage of the use of IL microemulsions is the possibility of reuse of the entrapped enzymes, something that is not feasible in other microheterogeneous systems. Immobilization of lipases which are entrapped in IL microemulsions in gels prepared with (hydroxypropyl)methyl cellulose (HPMC) confers the improved catalytic features that the lipases exhibit in these microemulsions available to various non-conventional media. The immobilized lipase in IL microemulsion-based organogels led to increased synthetic activity, independently of the reaction medium. Moreover, the stability of immobilized lipases is increased compared with that exhibited by non-immobilized enzymes in an aqueous solution (up to 200 times) or IL microemulsions (up to 25 times). The increased stability of lipases in microemulsion-based organogels is due to the adoption of a more rigid structure, as in the case of IL microemulsions. Functionalized multi-walled carbon nanotubes (CNTs) and graphene oxide derivatives which bear chemical groups (such as carboxyl-, hydroxyl- and amino-groups) or aliphatic chains on their surface were used as immobilization carriers for lipases and esterases. Enzymes can be immobilized onto these nanomaterials with physical adsorption and covalent attachment, producing biomaterials that can be loaded with up to 1.65 mg enzyme per mg of nanomaterial. The immobilization yield and catalytic activity of enzymes is greatly affected by the geometry and the functional groups of the nanomaterials and the immobilization protocol. The experimental results suggest the functionalized CNTs as more appropriate immobilization carriers than the respective graphene oxide derivatives. Regarding the functional groups on the surface of the nanomaterials, carboxyl-groups seem to be recognized by the active site of hydrolytic enzymes, leading to reduced activity, while further modification of these nanomaterials with hexamethylenediamine have a positive effect on the catalytic activity of enzymes. The synthetic

ix

Abstract

activity of immobilized hydrolases is up to 75 times greater than that of free enzymes. The covalent immobilization of enzymes producing materials with comparable biocatalytic activity to the physical absorption protocol and leads to significant stabilization of enzymes. The enzyme - nanomaterial interactions lead to structural changes of enzymes, particularly during non-covalent immobilization. The presence of nanomaterials in aqueous solution leads to reduction of enzymes -helical content. This reduction has a negative impact on the catalytic activity of esterases and while for lipases it led to activation. This can be related to the interfacial activation of lipases. It seems that organized nanostructures (either microemulsions or nanomaterials) simulate in a better way the natural microenvironment of these enzymes compared with the aqueous solution, resulting in a structure of the enzyme observed in these systems which is closer to the active structure of lipases when they act in nature. These novel nanobiocatalytic systems can be used to develop biocatalytic processes, such as modification of bioactive natural compounds and the production of biodiesel. Conclusively, organized nanostructured systems significantly improve the catalytic activity of hydrolytic enzymes, leading to novel nanobiocatalytic systems with interesting properties. The systems developed in the present study are consistent with the requirements of 'green chemistry', as their structural components exhibit low toxicity and high biocompatibility. The results of this study demonstrate the significant benefits arising from the implementation of organized nanostructures to entrapment and immobilization of hydrolytic enzymes for the development of novel innovative biocatalytic systems, which constitute the basis for the development of numerous applications in the field of nanobiocatalysis and, in a more wide perspective, in the field of nanobiotechnology.

x

&

& : [Bdmim]BF4 1,3--3- [Bmim]BF4 [Bmim]PF6 AOT ATR Bs2 CalA CalB CI CNT CNT-NH CNT-R Crl CTAB Cvl F FTIR F GC Gr-COOH Gr-NH Gtl HPLC HPMC IL NCI PestE Pfe I pNPB pNPP r r2

1--3- 1--3- -(2-) Bacillus subtilis A Pseudozyma ( Candida) antarctica Pseudozyma ( Candida) antarctica ( ) Candida rugosa - Chromobacterium viscosum Fourier , Geobacillum thermoleovorans () Pyrobaculum calidifontis Pseudomonas fluorescens 4- (C16) 4-

CNT-COOH

RM

xi

&

Rml RMSD SDS SLM t1/2 TEOS Tll TMMS TMOS TMPS Tris Tween 20 wo XPS

Rhizomucor miehei Thermomyces lanuginosa (o) (o) 2-A-2-()-1,3- M --

Triton X-100 p-(1,1,3,3-oo)

: max ( ) ( )

: Novozyme 435 2,2,4- cis-9- 1,2,3- B Pseudozyma antarctica

xii

1

1 1.1 1.21.2.1 1.2.2 1.2.3

7 7 1113 14 16

2 2.1 2.22.2.1 2.2.2 2.2.3

-

18 18 2121 23 24

2.3 2.4 3 3.1 3.2 4 4.1 4.2 4.3 4.4 4.54.5.1 4.5.2

O -

25 28 30 30 32 35 35 36 39 41 4242 45

5 5.1 5.2

5.2.1 5.2.2 5.2.3

48 48 5051 55 57

5.3

-

58

xiii

6 6.16.1.1 6.1.2 6.1.3 6.1.4 6.1.5

/

63 6363 64 64 65 65

7 7.1 7.2 7.3 7.4

7.2.1 7.2.2

66 66 6767 67

67 6969 70 71

7.4.1 7.4.2 7.4.3

7.5 7.6

7.5.1 7.5.2 7.6.1 7.6.2 7.6.3 7.6.4 7.6.5 7.6.6

72 74

72 72 74 74 75 75 76 77 77 78 78 79 79 79

4- - ()1-

7.7

7.7.1 7.7.2 7.7.3 7.7.4 7.7.5 7.7.6

77

7.8 7.9

7.8.1 7.8.2

(GC) (HPLC)

81 82

81 82 82 83 83 83 84 84

7.9.1 7.9.2 (CD) 7.9.2.1 7.9.2.2 7.9.2.3 7.9.3 (FTIR)

xiv

7.9.3.1 7.9.3.2 7.9.3.3

84 85 85

7.10

7.10.1 7.10.1.1 7.10.1.2 7.10.2 7.10.2.1 (FTIR) 7.10.2.2 Raman 7.10.2.3 (XPS)

&

87

87 87 89 89 89 89 90 90 90

7.11

7.11.1 7.11.2 -

90

8 8.1 8.28.1.1 8.1.2 8.2.1 8.2.2

-

95 9696 98 99

99

101 104 107 109 111 113 114 117 120 122 126

8.3

8.3.1 8.3.2 8.3.3 8.3.4 8.3.5

103

8.48.4.1 8.4.2 8.4.3

114

8.58.5.1 8.5.2

121

8.6 9 9.1

128 132 133

9.1.1 9.1.2 9.1.3

HPMC

134 135 136

xv

9.29.2.1 9.2.2 9.2.3 9.2.4

137138 139 142 143

9.39.3.1 9.3.2

144144 147

9.4 9.5 9.6

9.5.1 9.5.2

HPMC

148 149 153 156 157

149 151

10 10.1 10.2 10.3 10.4

10.1.1 10.1.2 Raman

157 159

10.4.1 10.4.2

161 164 167

167 171

11 11.1

11.1.1 11.1.2 11.1.3 11.1.4

175 175

175 177 178 179

11.2 11.3 11.4

11.4.1 CNTs 11.4.2 11.4.3

XPS

181 183 186

186 187 189

11.5 11.6 11.7 12 13

CALB

190 193 195 197 205 231

(-IV)

xvi

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2

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9

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11

1995, Ollis et al. 1992). . (Verger 1997), - (Chahinian et al. 2002, Levisson et al. 2009).

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12

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, , / (Schrag and Cygler 1997). - , 8 - (1-8) , - (Pleiss et al. 1998). / 3. . , (Ser), (His) (Asp) (Glu) (Jaeger et al. 1999, Neves Petersen et al. 2001, Schrag and Cygler 1997). Gly-X-Ser-X-Gly (Gly ), - 5 C ( 3). , . , , . -, (Jaeger et al. 1999, Pleiss et al. 1998). ( )

13

(Brady et al. 1990, Sarda and Desnuelle 1958). , (.. Bacillus subtilis) - (.. Pseudozyma antarctica) (Uppenberg et al. 1994, van Pouderoyen et al. 2001).

3: () / . - -. () Pseudozyma antarctica (CalB, pdb 1TCA) . .

, , . - (Hari Krishna and Karanth 2002, Paiva et al. 2000). , . 1.2.2 . . ,

14

-, . , Ser-His-Asp/Glu, , . 2 ( 4).

4: . (Asp) , .

( 4-). , ( 4-). .

15

, , (Asp/Glu), , . , . ( 4-). , . , . (-) . , , . , . (Bornscheuer and Kazlauskas 2004, Cygler et al. 1994, Jaeger et al. 1999). 1.2.3 (Martinelle et al. 1995, Verger 1997). . , : (Hari Krishna and Karanth 2002). , Brockman (Brockman et al. 1973). . , (Entressangles and Desnuelle 1974, Hari Krishna and Karanth 2002, Silber et al. 1999). , Rhizomucor miehei 1990 () (Brady et al. 1990, Winkler et al. 1990). o ,

16

. , . , , , , (Paiva et al. 2000). (Brzozowski et al. 1991, Marangoni and Rousseau 1995) (Overbeeke et al. 2000, Verger 1997). , 1958, , , (Sarda and Desnuelle 1958). . , Pseudozyma (Candida) antarctica Pseudomonas glumae Pseudomonas aeruginosa (Jaeger et al. 1994, Verger 1997). , (Hari Krishna and Karanth 2002, Jaeger and Reetz 1998).

17

2 2.1 - 1 , . , . , . 2 . , , ( 5). . , , . (Fanun 2008, Sottmann and Stubenrauch 2009).

5: .1 2

.. . .

18

. . (.. Tween 20 3, Triton X-100 4) . (.. AOT 5), (.. CTAB 6) . (critical micellar concentration, CMC) . . , ( 6).

6: - - . ( ) ( ).

, 6. 3 4

M -- -(1,1,3,3-) 5 -(2-) 6 -

19

, . , . : . . , , (Danielsson and Lindman 1981). (Fanun 2008, Sottmann and Stubenrauch 2009). m. , 1.5 - 100 nm. -, . . 6 , ( 6). (oil in water, o/w). , ( 6). (water in oil, w/o) , . ( ) , (Fanun 2008). . (wo) ( 1), (Carvalho and Cabral 2000). { }

7.

20

. . , . , ( ) (Carvalho and Cabral 2000).

7: .

, -. (Sottmann and Stubenrauch 2009). , (De Gennes and Taupin 1982).

2.2 2.2.1 .

21

CTAB, , . () n-, n- . / / , . -(2-) , Aerosol OT ( 5), , n- (Zulauf and Eicke 1979). ( 8% v/v), (Luisi et al. 1988, Luisi and Magid 1986). . 15 100 , , (Luisi et al. 1988). , , , , (Carvalho and Cabral 2000, Klyachko and Levashov 2003, O ch Sch mck 2002), (D s B.N y 2006, Eastoe et al. 2006, Husein and Nassar 2008, Turco Liveri 2006).

8: .

22

(Luisi et al. 1988). , 10-7 s, (Martinek et al. 1986). . , ( 8) (Luisi et al. 1988, Luisi and Magid 1986). 10-4 s , (Fletcher et al. 1987). 2.2.2 (.. ) -. , . , . , , . (Lund and Holt 1980, Smith et al. 1977). -, . (Khmelnitsky et al. 1988a, Tzialla et al. 2008, Tzialla et al. 2009). 2: .

/ / 2- n- / / 1- n- / / 1- - D- / / - / / 2-

(Lund and Holt 1980) (Topakas et al. 2005) (Topakas et al. 2003a) (Tzialla et al. 2009) (Tzialla et al. 2008)

n- / / 2- (Smith et al.

23

1977), . , 2. , / / (Voutsas et al. 1999). 2.2.3 , . ( 9), 100 C.

9: .

, (Kragl et al. 2002, Park and Kazlauskas 2003, Sheldon et al. 2002, van Rantwijk and Sheldon 2007), (Sheldon 2007, van Rantwijk and Sheldon 2007). , , . . , .

24

. 3. 2004 (Gao et al. 2004), (Moniruzzaman et al. 2010c, Qiu and Texter 2008, Zhao 2010) (Moniruzzaman et al. 2008b, Xue et al. 2011) (Moniruzzaman et al. 2010b). 3: .

[bmim] PF6 7 / / Tween 20 [bmim] PF6 / / Triton X-100 [bmim] PF6 / / [omim] Tf2N 8 / / / 1- [bmim]PF6 / / / Triton X-100

(Gao et al. 2006) (Gao et al. 2005) (Shu et al. 2008) (Moniruzzaman et al. 2008a) (Xue et al. 2011)

2.3 , . . . , - , . , , -. 1977, Martinek - , (Martinek et al. 1977). , 7 8

1--3- 1--3- ()

25

(Luisi et al. 1977). , . -

. , , (Stamatis et al. 1999). , . , . (O ch Stamatis et al. 1999): - -, - - , (in vivo) . , (Avramiotis et al. 1996, Svensson et al. 1996, Walde et al. 1990). . . , . (Hari Krishna and Karanth 2002, Khmelnitsky et al. 1988b). Sch mck 2002,

26

, (Zaks and Klibanov 1988). (wo). . , wo - . , , , . ( ) -, (O ch Sch mck 2002).

(Luisi et al. 1988, Stamatis et al. 1993). , - . , , , (Stamatis et al. 1999). 10 .

10: - (), () ().

. ,

27

(

s

1998, Liu et al. 2000, Rees and

Robinson 1995). , (Orlich and Sch mck 2002, Stamatis et al. 1999). , . , pH, wo (Valis et al. 1992).

2.4 o , , , . 4 . , , . 4: .

- ,

, -Brij30, n- , , , CTAB - 1-, n- ,

(Jourquin and Kauffmann 1998)

(Barbaric and Luisi 1981) (Rodakiewicz-Nowak et al. 2005) (Macris et al. 1996) (Ayyagari and John 1995)

ibuprofen

(Hilhorst et al. 1983)

(Karayigitoglu et al. 1995)

28

, , , , , ... , . (Rodakiewicz-Nowak et al. 2005), , ibuprofen (Ayyagari and John 1995). , , 2 (Kurganov et al. 1985) , (Jourquin and Kauffmann 1998). . . , . , . (Hayes and Marchio 1998, Noritomi et al. 2008, Watarai 1997), (Shu et al. 2008). 2008 (Zhang et al. 2008). (Moniruzzaman et al. 2008b), (Xue et al. 2011), (Moniruzzaman et al. 2009, Zhou et al. 2008) (Zhang et al. 2008). , . , , , . .

29

3 3.1 - , , . . . , . . , (Luisi et al. 1990, Zoumpanioti et al. 2010). (gels) : . (hydrogels), (organogels)(Zoumpanioti et al. 2010). 1986 (Haering and Luisi 1986, Quellet and Eicke 1986), . . . , , . (Capitani et al. 1988, Capitani et al. 1991), (Zhao et al. 2006), (Atkinson et al. 1991, Petit et al. 1991), (Petit et al. 1991), (Quellet et al. 1991). , 11:

30

11: . ,, .

. . . . . . , . . , . (Luisi et al. 1990, Trickett and Eastoe 2008, Zhao et al. 2006), (Delimitsou et al. 2002, Feng et al. 2009, Pastou et al. 2000b, Stamatis and Xenakis 1999), (Feng et al. 2009, Stamatis and Xenakis 1999) (Delimitsou et al. 2002, Pastou et al. 2000b, Zoumpanioti et al. 2008). , .

31

: , . . . 30

C.

, . : D-, (1,4) - . , , . , . , . . , , . () (HPMC), , . , . () , (Li et al. 2005a, Siepmann and Peppas 2001), (Burdock 2007) (Doughty and Glavin 2009)

3.2 . , , (Backlund et al. 1996, Rees et al. 1991). Nascimento (Da Graca Nascimento et al. 1992). , , Chromobacterium viscosum Candida rugosa. , ,

32

CTAB . 5 . . 5: .

Candida rugosa

HPMC

CTAB AOT, CTAB Triton X-100 , AOT AOT (D m u D M

. 2004)

(Lopez et al. 2006) (Dandavate and Madamwar 2008) (Dave and Madamwar 2008) (Backlund et al. 1996) (Nagayama et al. 2003) (Rees et al. 1995)

Chromobacterium viscosum, Candida sp. Rhizopus delemar Chromobacterium viscosum Trichosporon capitatum Rhizomucor miehei, Candida antarctica () Pseudomonas cepacia Rhizomucor miehei

AOT

(Song et al. 2008)

, HPMC , , HPMC

, ,

(Pastou et al. 2000b) (Xenakis and Stamatis 1999) (Zoumpanioti et al. 2008)

, 10-14 % (w/v). , (Zoumpanioti et al. 2010). , . , .

33

, . , . , (Delimitsou et al. 2002). HPMC 50 C, (Zoumpanioti et al. 2008). , , , , . Schuleit Luisi (Schuleit and Luisi 2001). , , .

34

4 4.1 - , . 1 100 nm. , , . (Kroto et al. 1985) (Iijima 1991) . (Kuchibhatla et al. 2007, Whitesides 2005): , . , . , , . , . . , (: (Pagliaro 2010, Schmid 2010, Whitesides 2005)), . , , , (Kuchibhatla et al. 2007, Whitesides 2005). , (Kuchibhatla et al. 2007). , . , , , , , , . 6 . , ,

35

( ). 6: .

(Ravelo-P

(Ji et al. 2010) . 2010)

(Fujigaya et al. 2010) (Upadhyayula et al. 2009) (Foldvari and Bagonluri 2008) (Shipway et al. 2000) (Brayner 2006) (Ding et al. 2010) (Pauliac-Vaujour et al. 2011) (Bosi et al. 2003) (Babu et al. 2010) (Ghosh et al. 2008) (Liang et al. 2009)

,

4.2 , . , , . , . , , (Hennrich et al. 2006). . 6 , 1s, 2s 2p. 4 , 2s 2p. 2s 2p , . p , sp1, sp2 sp3 . sp3 (Hennrich et al. 2006).

36

: , (Hennrich et al. 2006, Mostofizadeh et al. 2011). , 0 3 . 12.

12: () , () C60, () , (V) ( ).

sp3 4 . . sp3 , (Hennrich et al. 2006). sp2 , , . . , , . , . p , -. - . (Park and Ruoff 2009, Tung et al. 2009). . sp2 , . , ( ) . , 4.4 (Bosi et al. 2003, Hennrich et al. 2006). , , .

37

1985 Smalley (Kroto et al. 1985). , 1991 , Iijima (2-20 ) (Iijima 1991). (Bethune et al. 1993, Iijima and Ichihashi 1993). Iijima, 1960 Bacon, o , (Bacon 1960). (carbon nanotubes, CNTs) , , (Ajayan and Tour 2007, W . 1998). ( ), ( ), 13. 100 nm, (Capek 2009). (Dyke and Tour 2006) , (Chamberlain et al. 2010, Tasis et al. 2006). , , , (Baddour and Briens 2005, Kumar and Ando 2010).

I

II

III

IV 13: (-) (IV). (), - () ().

38

0.4 3 nm. 0.14 nm, , (Bonard et al. 2001). , - ( 13 13 ). , ( 13). , . ( 13IV) 100 nm, 2 20 . 0.340.36 nm, (Capek 2009). , -, (Kuchibhatla et al. 2007).

4.3 . , . , . , van der Waals ( M 2010). , . , , (Dyke and Tour 2006). , . sp2 sp3 , , ( M 2010). .

39

. . -, - - , . (Asuri et al. 2007, Asuri et al. 2006a, Capek 2009), , (Bourlinos et al. 2003, Zhang et al. 2010b). . , (Georgakilas et al. 2008). , (Tasis et al. 2006). , . : () , () (Hauke and Hirsch 2010, Tasis et al. 2006). , , , , , , (Dyke and Tour 2006, Tasis et al. 2006). , (Kuchibhatla et al. 2007, Shim et al. 2002). , , (Karakoti et al. 2006, Sohaebuddin et al. 2010), (Bianco et al. 2005). , ( , ..) (Karakoti et al. 2006). (Jia et al. 2005).

40

, (Bianco et al. 2005, Kuchibhatla et al. 2007, Shim et al. 2002). , (Shim et al. 2002), (.. , ). . , , - (Thomas 2009), (Cirillo et al. 2011, Iancu et al. 2011), (Lu and Liu 2009) (Mitchell et al. 2002).

4.4 . 1992 6 carbon nanotubes 2010 8000 ( Web of Science ISI). : 1996 Smalley (Kroto et al. 1985), 2010 Geim Novoselov (Geim and Novoselov 2007). , . , sp2 , -. . , , , (Capek 2009). , (Hennrich et al. 2006), . , sp2 . . .

41

, (Treacy et al. 1996). , , . , , (De Heer 2002). sp2 0.14 nm, . sp3 , (Yu 2004). . . , (Bornscheuer 2003, Gao and Kyratzis 2008, Kim et al. 2006a). , , (Capek 2009). , . , (Bianco et al. 2005). .

4.5 4.5.1 1990 , . , (Kim et al. 2008). , ( 1.3 mg mg ) (Asuri et al. 2007). ,

42

. , (.. ) , (tailor-made). , , (Du et al. 2007b, Lin et al. 2009).

. , . , (Gao and Kyratzis 2008). , ( m . 2005, Shim et al. 2002). (- ). , . . , , , (Gao and Kyratzis 2008). Azamian (Azamian et al. 2002). , , (Matsuura et al. 2006). , . , , . , (Gao and Kyratzis 2008). ,

43

, . - (Asuri et al. 2007, Chen et al. 2009, Li et al. 2005b). . , , 1--3-(3) (EDC), (Gao and Kyratzis 2008, Lin et al. 2004). . Azamian EDC (Azamian et al. 2002). EDC, , . 1- (Chen et al. 2001). - , . 1 , . . , . (PEG) (Lin et al. 2004, Shim et al. 2002), , (Foldvari and Bagonluri 2008). . , . CTAB (Wang et al. 2009), Triton X-100 (Azamian et al. 2002, Shim et al. 2002) Tween 20 (Chen et al. 2003). Tween 20 ,

44

, (Chen et al. 2003). . , , , , . 4.5.2 , , , . , , , (Park and Ruoff 2009, Tung et al. 2009). , . . , (A . 2008). , , DNA (Foldvari and Bagonluri 2008). (A Y -S . 2008, Arya et al. 2008, Shao et al. 2010, . 2010). , . 2008, Arya et al. 2008).

, P450scc (A

. (Kim et al. 2008, Kim et al. 2006b). .

45

. , (Du et al. 2007b, Kim et al. 2008, Kim et al. 2006b). . , , (Bianco et al. 2005, Foldvari and Bagonluri 2008). (Bianco et al. 2005). , , , . 7 .

46

7: .

H2O2 RNA

(Portaccio et al. 2006) (Boland et al. 2009) (Pizzariello et al. 2001) (Wu et al. 2010a) (Wu et al. 2010b) (Wang et al. 2011) (Dey and Raj 2010) (Yang et al. 2010) (Song et al. 2011) (Zhang et al. 2010a) (Zhang et al. 2010b) (Liu and Cai 2007) (Yin et al. 2007) (Zhang and Zheng 2008) (Chen et al. 2009) (Smolander et al. 2008) (Liu et al. 2006) (Shah and Gupta 2008) (Jiang et al. 2009) ( m . 2005)

C

C C C - C

(Shi et al. 2007) (Ahuja et al. 2011) (Yi et al. 2008) (D'Souza et al. 2005) (Chen et al. 2007) (Chuang and Shih 2001) (Nednoor et al. 2004)

47

5 5.1 , 9, . , . , , ( 1.2). ( ) , , . , , . L- . () , . 14. , , .

14: 2 .

. 9

(.. ) .

48

(van Holde et al. 2006). . , . . : () , (), () (Fersht 1999). - - (-) . . .

15: () . () Ramachandran. .

, . 2 . - , -

49

( 15). . +180

-180 ,

. Ramachandran ( 15). Ramachandran, . - , (Fersht 1999). 4 , .

5.2 , . , ngstr m, (.. ), . . , , , . , . , 30 KDa, (Clore and Gronenborn 1998). . (Kelly and Price 2000). , , , . in silico (Katsoura et al. 2009, Otto et al. 2000).

50

. , (Gorke et al. 2010, Kragl et al. 2002, Park and Kazlauskas 2003, Sheldon et al. 2002, van Rantwijk et al. 2003, van Rantwijk and Sheldon 2007). , (Kelly et al. 2005). , in situ, . , 4 -, . , . (150 nm - 25 m). , , , (Van Mierlo et al. 2000). (Circular Dichroism - CD), (Fourier Transform Infrared FTIR) . in situ , , (Ganesan et al. 2009). 5.2.1 ( ) ( 16). , . , , , , .

51

( 16), ( ) (Berova et al. 2007).

16: () . () (van Mierlo et al. 2000).

. . , , , , . , , (Kelly and Price 2000).

17: (Kelly et al. 2005).

52

(180 250 nm). , Ramachandran, . , 17. , - 222 nm 208 nm 190 nm , - 218 nm 195-200 nm. , . (Greenfield 1996, Greenfield 2004, Van Mierlo et al. 2000). . SELCON (Sreerama and Woody 1993), VARSLC (Manavalan and Johnson Jr 1987), CONTIN (P ch ckner 1981) K2D (Andrade et al. 1993), , , (Greenfield 2004).

18: . (Ranjbar and Gill 2009).

53

, . (250-350 nm), . 18. , , . , . , (Kelly and Price 2000). c, , . , (Blauer et al. 1993, Santucci and Ascoli 1997). , Soret, , . (Berova et al. 2007, Kelly and Price 2000). , , . , , . , . , . , , (Berova et al. 2007, Kelly and Price 2000). , , .

54

5.2.2

, , . . , , . , (, , ) (Barth 2007). , , . , , , (Barth 2007, Jackson and Mantsch 1995). (A 1999, Cooper and Knutson 1995, Goormaghtigh et al. 1994a, Goormaghtigh et al. 1994b, Jung 2000, Manning 2005, Surewicz et al. 1993), (Barth and Zscherp 2002, Breton 2001, Mantele 1993, Marshall and Rich 2009), (Ganim et al. 2008, Lewandowska et al. 2010, Seshadri et al. 2009, Tantipolphan et al. 2008). , (Barth 2007), . , . 1950, , (Elliott and Ambrose 1950). , , Ramachandran (Mirkin and Krimm 2002). . 9

55

( VII, ) (Barth 2007, Barth and Zscherp 2002). , , 8. . , . 8: .

(cm-1) 1600 1700

(, %) C=O (80%) C-N (20%)

1500 1600

- (60%) C-N (40%) C-C (10%)

1200 1350

N-H C=O C-N C-C ( )

, . , , . , , (Jackson and Mantsch 1995). (Arrondo et al. 1993, Cai and Singh 1999, Jackson and Mantsch 1995). (Chittur 1998, Dong et al. 1990). . , (Cai and Singh 1999). , , , 10 (Barth 2007). .

56

. , . , (Forato et al. 1998), , (Chittur 1998). 5.2.3 , . , . ( 1-10 nsec), , . . , . 19.

19: . (1) , (2) , (3) .

57

, , , . 280 nm . , , 295 nm (Eftink 1991, Eftink 1998). , . . , , . , . (F) (max) (Eftink 1998, Monsellier and Bedouelle 2005). , 350 nm, 308 nm . . , (Eftink 1994). , ( ), , (Dumoulin et al. 2002, Monsellier and Bedouelle 2005, Mu et al. 2008, Ruiz et al. 2003).

5.3 - , , ( , ...) (Moniruzzaman et al. 2010c, Pavlidis et al. 2010, Stamatis et al. 1999, van Rantwijk and Sheldon 2007, Zoumpanioti et al. 2010). , .

58

in situ , . , . De Diego Candida antarctica (De Diego et al. 2005). , (Lau et al. 2004). , , (Mukherjee et al. 2007, Palomo et al. 2003). , (Marangoni 1993, Walde 1993). . , , . . (Karajanagi et al. 2004, Matsuura et al. 2006, Yi et al. 2008), (Asuri et al. 2006a, Chen et al. 2009, Du et al. 2007b). , (Jiang et al. 2004). , , , (Mu et al. 2008). - . , .

59

60

&

6 6.1 6.1.1

: 1. Pseudozyma ( Candida) antarctica (CalA). : 2.04 U/m ( uk ) 22.4 m /mL (Novozyme 735, Novozymes) 2. Pseudozyma ( Candida) antarctica (CalB). : 10.8 U/m 13.2 % w/w ( uk ) 11.9 m /mL (L Novozymes) A (N ym 435, N ym s) 3. Candida rugosa (Crl). : 2.9 U/m 2.6 % w/w (S m ) 724 U/m 8.4% w/w (Sigma) 4. Chromobacterium viscosum (Cvl). 3040 U/m 55.7 % w/w (B Ch m c ) 5. Rhizomucor miehei (Rml). 0.92 U/mg (Fluka) (14.0 % w/w ) 6. Thermomyces lanuginosa (Tll). 34.7 mg/mL (L ym TL 100 L, N ym s) ym CALB L,

Greifswald (), Escherichia coli. 1. Bacillus subtilis (Bs2). 28.8 % w/w 2. Pyrobaculum calidifontis (PestE). 82.5 % w/w 3. Pseudomonas fluorescens (Pfe I). 479 g/mL 4. Geobacillus thermoleovorans (Gtl). 60.5 % w/w (Sigma) , .

63

6.1.2

M ck, Sigma, Fluka, Pareac, Riedel de Hen (>99 %). : 2 18 , (C18:1). : (C4) (C16) 4- (pNPB pNPP ) . , . : 1 8 , ()1-. :

2- , 3- , , , - , , . 6.1.3 (, , ) Fluka Riedel de Hen. 50 m, pH 7.5. Tris HCl, , Tris . M ck, P c L bSc . (>97%): [bmim]BF4: 1--3- (A ch) I )

[bmim]PF6: 1--3- (A h s ) [bdmim]BF4: 1--2,3- (S

64

6.1.4

. , () (HPMC) 10 Sigma. , Fluka, 20.

20: .

. (CNTs) Aldrich 2-6 nm, 10-15 nm 0.1-10 m, Fluka. , 7.10.1. 6.1.5 / -(2) () p-(1,1,3,3) (Triton X-100) Sigma. -- (Tween 20) Fluka. ( 7.3) Sigma Fluka, Fermentas. 3 4 Sigma Fluka. .10

2 % (w/v) 40 - 5600 cP 20 C.

65

7 7.1 3 . . Bradford: Coomassie Brilliant Blue G-250 (Bradford 1976). 465 nm 595 nm. 900 L Bradford 100 L (