Novel Sanger's Reagent-like Styrene Polymer for the Immobilization of

Subscriber access provided by University of South Dakota

Biological and Medical Applications of Materials and Interfaces

A Novel Sanger’s Reagent-like Styrene Polymer for the Immobilization of Burkholderia cepacia Lipase Junying Fu, Zhiyuan Wang, Wen Luo, Shiyou Xing, Pengmei Lv, Zhongming Wang, and Zhenhong Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09225 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

A Novel Sanger’s Reagent-like Styrene Polymer for the Immobilization of Burkholderia cepacia Lipase Junying Fu †,

, Zhiyuan Wang †,

‡, §

‡, §*

, Wen Luo †,

, Shiyou Xing †,

‡, §

‡, §

, Pengmei Lv †,

‡, §, ¶**

,

Zhongming Wang†, ‡, §, Zhenhong Yuan†, ‡, §, ¶ †

Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China ‡

Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, China §

Guangdong Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, China

¶

Collaborative Innovation Center of Biomass Energy, Henan Province, Zhengzhou 450002, China

Keywords: Sanger’s reagent; Immobilization; Lipase; Hydrolysis; Enantioselectivity

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

Abstract: Sanger’s reaction, which was originally developed for amino acid detection, was utilized for enzyme immobilization. The newly synthesized polymer support, which was called pNFD, was embedded with a Sanger’s reagent-like functional group for immobilizing enzymes covalently under mild reaction conditions. Using Burkholderia cepacia lipase (BCL) as the target enzyme, the immobilization efficiency and activity of pNFD-BCL reached as high as 1.2 mg·g-1 and 33.21 U·g-1 (specific activity of 27, 675 U·g-1) respectively, realizing a 90% activity recovery. It also improved the optimal reaction temperature of BCL from 40℃ to 65℃, under which its full-activity could be retained for 4h. The new carrier also widened the pH adaptive range of BCL as 6.5-10.0, allowing the lipase to operate normally at weak acid environment. Reusability of pNFD-BCL was significantly improved as almost no activity and/or enantioselectivity lost was observed in 200h of triglyceride hydrolysis reaction and 17 batches of (R,S)-1-phenylethanol resolution reaction.

1 Introduction Enzymes are being used as biocatalyst in diverse fields, such as environmental conservation, pharmaceutical engineering and the food industry, etc. Generally, enzymes can work under mild reaction conditions with low energy consumption. However, biocatalysts (or enzymes) are also facing a technological hitch such as being prone to activity loss, high cost and poor reusability. In many situations, enzymes need to be separated after reaction to avoid product contamination. This process is time and energy consuming and the recovered enzyme often has low residual activity. To overcome these disadvantages, many studies have been performed to afford enzymes with high reusability.1-2 Immobilization of enzyme on a stable support is one of these promising alternatives.3-4 Protein molecules immobilized on supports through physical interaction such as adsorption and trapping, for example, by taking advantage of a weak intermolecular force instead


2

Page 3 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of forming new chemical bonds between the enzyme and supports. Although the nature of enzyme can be largely preserved, it can easily leach out especially when the reaction is processed under vigorous stirring and end up with unsatisfactory reusability. Enzyme molecules are immobilized by introducing new chemical bonds which are stable enough to avoid enzyme leaching from the support.5-6 In both methods, the most important requirement is that the immobilizing process should not lead to target protein denaturation or deactivation. The functional groups of amino acid residues present on the surface of proteins are often used to bond to the support: such as the carboxyl, amidogen, guanidyl, sulfydryl and imidazole groups, among others. Among these groups, the ε-amine (ε-NH2) group from lysine (Lys) is a widely used choice for the following two reasons. First, hydrophilic Lys is preferentially distributed on proteins’ surface, which provides easy access for the connector on carriers.7 Second, Lys has a long side chain that largely maintains the conformational flexibility of the immobilized protein molecules and, in turn, their activity. In addition, the reactions used for ε-NH2 of lysine linking were amidation, diazotization, alkylation and the Schiff reaction,8-12 allowing wider support choices. In the well-known Sanger's reaction, 2, 4-dinitrofluorobenzene (DNFB), which is called Sanger’s reagent, reacts with -NH2 on amino acids in weak base solution at room temperature to produce dinitrophenyl amino acid (DNP-amino acids) through a electrophilic substitution of F on the aromatic ring. This reaction was developed by Sanger and used for amino acid detection (Scheme.1)13-14. Except for the α-NH2 from amino acids or from N-terminal amino acid residue of the protein, the ε-NH2 of lysine (or lysine residue in protein) can also react with Sanger's reagent (including the situation when the –NH2 is protonated, see Figure S1 and S2). If a support


3


Page 4 of 39

could be modified to have a DNFB-like functional group, proteins then could be immobilized on supports through Sanger reaction under mild condition. Scheme 1. Sanger’s reagent for amino acid detection

F + O 2N

NO2

Sanger‘s reagent DNFB

COOH H2N CH R

R H N CH COOH

Weak base solution O2N

amino acid

+

F-

NO2 DNP-amino acid

Based on this inspiration, we have successfully synthesized a new type of polymer applying the suspension polymerization method by introducing 4-fluorostyrene into the polymer framework to produce a new polymer, poly-(4-fluorostyrene-divinylbenzene) (FS-DVB). After nitration by fuming nitric acid, the new polymer had a DNFB-like structure, which resembled the chemical properties of DNFB, to provide an acceptor for ε-NH2 of the target protein. We called this functional polymer NO2-FS-DVB, abbreviated as NFD. The porous NFD (pNFD) with higher specific surface area was also synthesized to improve its immobilization capacity. In this research, we used Burkholderia cepacia lipase (BCL) as the model enzyme to test the immobilization ability of the newly synthesized carrier. With a c.a. 4.5 nm molecular size, it contains 320 amino acids residues, including a catalytic triad of Ser87, His286 and Asp264, which are mainly covered by hydrophobic residues at its close conformation. These residues constitute the ‘lid’ structure, enabling BCL to go through an interfacial activation at hydrophobic interface;15-16 when BCL molecule approaches the hydrophobic surface (or hydrophobic liquid phase), the conformation of the ‘lid’ structure, which is constituted of α4, α5 and α9 helixes, starts moving to expose its active center.15,

17

By taking advantage of this reaction, many

researchers have tried to immobilize BCL using hydrophobic carriers. During the interfacial activation, the covalent connection between BCL and the carrier is formed due to its closeness


4

Page 5 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with the amino acid residues on BCL, yielding and maintaining an ‘immobilized open structure’.18-20 In the polypeptide chain of BCL, there are eight potential residues, including seven Lys residues and the N-terminal alanine (N-Ala) residue, both of which are theoretically available for the covalent connection, distributed on the outer surface of the molecule. Due to their different function or interaction with the active sites, covalent connection through each one or more of them might afford the immobilized BCL with different activity recovery. To realize the site(s) oriented protein immobilization from the aspect of immobilizing material, Abaházi et al. had tried by adding hydrophilic cross-linker while immobilizing BCL on hydrophobic surface and realized an enhanced racemization activity of lipase. However, its specific activity recovery was only 5-15%.21 BCL has a widely tolerance for several kinds of organic solvents such as propanol, toluene and ether, etc.22-25 Owing to its high enantioselectivity, it was often applied in the some non-aqueous racemic resolution reactions. The transesterification resolution of (R,S)-1-phenylethanol via vinyl acetate was one of the most adopted one on the immobilization research of BCL because similar reactions had been commercially applied in the production of important medical intermediates.2630

Besides, hydrolysis is one of the featured activity of lipase and it was often involved in the

reaction to produce biodiesel from triglycerides.31-32 To assess the applicability of pNFD-BCL immobilization enzyme, its catalytic property, and thermal stability were studied compared with those of the free state BCL and chitosan immobilized BCL. Based on triglycerides hydrolysis and resolution of (R,S)-1-phenylethanol, continuous batches of reactions were conducted to test the reusability and storage stability of pNFD-BCL.


5


Page 6 of 39

2 Experimental 4-Fluorostyrene (FS, 97%) and o-divinyl benzene (DVB, 80%) were employed to prepare a polymer carrier via the suspension polymerization method using benzoperoxide (BPO) as the initiator. Liquid paraffin and dibutyl phthalate (both in analytical grade) were used as porogens to produce porous polymer supports. Fuming nitric acid used for nitrating the polymer carrier was produced in the laboratory by distilling a 1:1 (v/v) mixture of concentrated sulfuric acid and 65% (w/w) nitric acid at 100℃ under vacuum. Burkholderia cepacia lipase (BCL, with cyclodextrin as support) was purchased from Amano Enzyme, Inc., Japan. Rapeseeds oil (RSO) was purchased at a local supermarket. (R,S)-1-phenylethanol and vinyl acetate were purchased from Sigma-Aldrich with purity higher than 99%. Other chemicals, such as the co-dispersant polyvinyl alcohol (PVA, alcoholysis degree 98%), and gelatin used in suspension polymerization process, p-nitrophenyl palmitate (pNpp) and Triton X-100 for enzyme activity analysis, were of analytical grade and used without further purification. 2.1 Synthesis of the FS-DVB polymer The scheme for producing this DNFB-like polymer is shown in Scheme 2 with the initiation of 4-fluorostyrene (FS) polymerization. The porous NFD support (pNFD) was synthesized by adding porogens during the polymerization step. After nitration with fuming nitrite acid, the functional polymer was applied for lipase immobilization. Scheme 2. Proposed route of the (p)NFD support synthesis and immobilization


6

Page 7 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HC CH2 +

Polymerization

O 2N

Nitration

NO2

F

F FS

F

Fuming nitric acid/ H2SO4

H O O H H H H N C C N C C N R CH2 CH2 CH2 CH2 OR NH2

Protein with Lys-residual on its surface

NO 2-FS-DVB (NFD)

FS-DVB Polymer

DVB

H O H H H N C C N C NH2 R R

N-terminal of protein

Immobilization pH7.8-8.0 PBS

H O O H H H H N C C N C C N R CH2 CH2 CH2 CH2 NH

O2N

NO 2

Main framework of FS-DVB

H O H H H N C C N C NH R R OR

O 2N

NO2

NO2-FS-DVB-Protein the immobilized enzyme

The dispersant solution was prepared by mixing 0.45 g PVA and 0.45 g gelatin into 90 mL of deionized water. The oil phase, including 9.00 g of FS and 1.00 g of DVB, was evenly mixed before adding BPO. To prepare the porous FS-DVB carrier, porogen was also introduced by adding 5.00 g of dibutyl phthalate and 4.00 g of liquid paraffin to the aforementioned oil phase. The dispersant solution was pre-heated in a 70℃ oil bath for 30 min before stirring with oil phase at 150-160 rpm (5 cm diameter agitator blade) in a 250 mL three-necked flask. The mixture was heated at 70℃ for 1 h and then at 80℃ for 2 h before accelerating the stirring rate to 200 rpm. After 4 h of continuous polymerization at 90℃, the white spherical polymer was filtered and washed with ethanol in ultrasonic cleanser to remove the unreacted FS, DVB and porogen. The dried product was stored in dry cabinet at room temperature before use. 2.2 Nitration of FS-DVB Nitration of FS-DVB was initiated by adding 2.00 mL of nitration acids (mixed by 3:1 v/v fuming nitric acid and concentrated sulfuric acid in ice bath) to every 1.00 g of FS-DVB (or pFSDVB). The nitration process lasted for 24 h at room temperature in the dark without stirring. The


7


Page 8 of 39

reaction was terminated by removing the extra fuming nitrite acid. The NFD (or pNFD) product was then washed by re-suspending it in 65% nitrite acid and gradually adding deionized water in ice bath until the washing solution became neutral. The product was then dried at room temperature and stored in a dry cabinet before use. 2.3 Enzyme Immobilization The NFD was first immersed in 50 mM pH 7.8-8.0 PBS (phosphate buffer solution, prepared by K2HPO4·3H2O and KH2PO4 with double deionized H2O, as followed) to balance its internal pH. Adding 2 mL BCL solution (2.5 %, w/w, in 50 mM pH 7.8-8.0 PBS, protein concentration [P] = 340 ± 10 µg·mL-1) to every 0.5 g of PBS-immersed NFD, the immobilization reaction was carried out in a 30℃, 200 rpm thermal shaker for 24 h. The immobilization capacity was determined by analyzing the protein concentrations of the original and residual enzyme solution using BCA method kit (Sangon Biotech Co. Ltd., Shanghai, China). The NFD-BCL or pNFDBCL product was dried at room temperature for less than 0.5 h, and stored at 4℃. 2.4 Characterization Nitrogen adsorption-desorption experiments were carried out to obtain the specific surface areas and pore width distribution according to the Brunauer-Emmett-Teller (BET) theory and BJH theory, respectively, with an American Quantachrome ASIQMO002-2 automated system at -196 ℃. The sample was outgassed at 100℃ for 16 h under vacuum prior to measurement. The microstructures of (p)FS-DVB, (p)NFD and (p)NFD-BCL were observed using HITACHI S4800 Field-Emission Scanning Electron Microscope (SEM). Fourier-transformed infrared spectroscopy (FT-IR) was also performed to verify the chemical connection between protein and the NFD. Samples for FT-IR analysis were prepared using KBr pellet method. 2.5 Enzyme Assay


8

Page 9 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lipase activities were measured based on the hydrolysis reaction of pNpp to produce pnitrophenol (pNp, Scheme 3), the concentration of which can be measured using the standard curve method by detecting the absorbance at 410 nm (A410). The unit activity is defined as the enzyme amount that can hydrolysis pNpp to produce 1 µmol pNp in 1 min. Scheme 3. Lipase catalyzed hydrolysis of pNpp O2N

O O

H2O 7

O2N

O +

Lipase

pNpp

OH

pNp

HO

7

palmitic acid

Enzymes are highly sensitive to ambient temperature compared to chemical catalysts. The effects of temperature on the hydrolytic activity of free lipase and immobilized lipase were tested from 30℃ to 80℃ in PBS pH 7.8-8.0. The pH adaptive range was measured in reaction buffer at pH from 4.0 to 10.0. Before the pNp concentration measurement, the pH of the terminated reaction solution was adjusted to its original pH condition (50 mM pH 7.8-8.0 PBS) by adding 0.5 mol·L-1 NaOH. For industrial application, the high-temperature endurance of lipase is of vital importance for long-term consecutive reactions. In this study, the thermal stability of free lipase and immobilized lipase was compared by first processing lipase at 65℃ for 0.5-8 h before testing its residual activity at 37℃. Km and Vmax can be calculated using the transformed Michaelis-Menten equation by building the linear relationship between 1/[S] ([S]: concentration of the substrates, µmol·L-1) and 1/[v] (rate of the substrates conversion under specified [S], µmol·L-1·min-1) (Equation 1). 1 Km 1 1 = · + v Vmax [S] Vmax

(Equation 1)

2.6 Oil hydrolysis by pNFD-BCL


9


Page 10 of 39

The triglycerides hydrolysis activity of pNFD-BCL was assessed using RSO as the substrate. This reaction was conducted in a 45℃, 200 rpm thermal shaker with reaction system set as 5 g RSO, 10 g deionized water and 0.5 g pNFD-BCL. The yield of fatty acids was analyzed using the same method used in our previous study.33 2.7 Resolution of (R,S)-1-phenylethanol by pNFD-BCL pNFD-BCL was also used to catalyze the resolution of (R,S)-1-phenylethanol using vinyl acetate as the acyl donor (Scheme 4). The molar ratio of (R,S)-1-phenylethanol and vinyl acetate was set at 4:1 using n-heptane as the solvent (1 mmol (R,S)-1-phenylethanol, 4 mmol vinyl acetate and 5.00 mL n-heptane, 100 mg pNFD-BCL). The reaction was carried out in a 37℃ thermal shaker at 200 rpm for 1 h. The reaction was terminated by simply removing the pNFDBCL particles. Meanwhile, 300 µL samples were withdrawn and filtered through a 0.22 µm membrane before dilution with 700 µL of n-hexane before analysis. The conversion and enantiomeric excess (ees%) were determined by a HPLC (Waters, USA, e2695 Separation Module with 2998 PDA detector) equipped with a Chiralcel® OD-H (φ4.6 mm×250 mm, 5 µm) chiral chromatographic column. The samples were eluted with a mixture of n-hexane/2-propanol (9:1, v/v) at 0.8 mL·min-1 for 12 min and detected at 254 nm.34 Scheme 4. BCL catalyzed resolution of (R,S)-1-phenylethanol O O OH CH3

CH3

CH3 +

O

O

Lipase CH2

O (R)-1-phenylethyl acetate

n-hexane (R,S)-1-phenylethanol

OH

vinyl acetate

CH3

+ H3 C

H

acetaldehyde

(S)-1-phenylethanol

2.8 Reusability test


10

Page 11 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The reusability of pNFD-BCL was tested and analyzed in the hydrolysis and resolution reaction using the same reaction conditions as aforementioned. Between each batch of reactions, if not specified, pNFD-BCL was simply filtered and directly used for the next batch of reactions without further treatment. 3 Results and Discussions 3.1 Chemical characterization of the (p)NFD-BCL 3.1.1 Immobilization performance of pNFD Based on the proposed synthesize routine shown in Scheme 2, the porous NFD (pNFD) support was prepared for BCL immobilization. The newly synthesized carrier, pNFD, showed an immobilization capacity (IMC) up to 1.2 mg·g-1 in a solution where the original BCL concentration was only 340 ± 10 µg·mL-1 (2 mL solution per 0.5 g dry carrier). Activity of pNFD-BCL (IMC=1.2 mg·g-1) varies from 8.44 U·g-1 to 33.21 U·g-1 (per gram of pNFD carrier, tested at 37℃, pH 7.8-8.0), corresponding specific enzyme activity from 7, 033 U·g-1 to 27, 675 U·g-1 (per gram of protein). Compared with the original specific enzyme activity of the free BCL, c.a. 30, 000 U·g-1, the activity recycling ratio reaches over 90% of its original. 3.1.2 Effect of BCL molecule orientation on performance of pNFD-BCL Previous results have shown that this poor reproducibility of the activity of the immobilization enzyme might be attributed to the aggregation of the open-conformation lipase molecules35 or the random amino acid residues connection with the carrier. After a simple calculation (see supporting information), we have precluded the former reason as there are large enough rooms for BCL immobilization without forming aggregates. Other reason, such as the immobilized close-conformation BCL also has probability of interfering the reproducibility, was also taken into consideration (see Figure S3). For the open-conformation BCL, we turned to believe that


11


Page 12 of 39

there must be preferred connection sites leading to better performance of the immobilized enzymes. According to the steric structure of BCL (PDB: 3LIP_A, three-dimensional molecular structure of the active-conformation BCL), there are seven Lys residues located on the surface of the BCL molecule, and their distribution is illustrated (red) in Figure 1A, 1B and 1C. N-terminal amino acid residue N-Ala (blue) was also taken into consideration. By comparing the exposed surface area shown in Figure 1D, we can presume that Lys22, Lys70, Lys165, Lys316 and N-Ala are more likely to complete this connection than Lys80, Lys269 and Lys283 because they are less influenced by the steric hindrance of the ambient residues, allowing a greater effective contact with pNFD.

Figure 1. Preferred amino acid residue connection sites analysis based on the steric molecular structure of BCL: (A)~(C), steric location of Lys residues and N-terminal amino acid residue;


12

Page 13 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(D), surface exposed area of Lys residues and N-terminal amino acid residue; (E), relationship between substrates accessibility and immobilization sites. The active center of BCL is cooperated by a trinity combination: Ser87, His286 and Asp264, the location of which is also shown in Figure 1C (yellow). By comparing its steric location and distances to Lys22, Lys70, Lys165, Lys316 and N-Ala respectively, it is clear that Lys316 and N-Ala locate farther from the active center than the other three residues. At close-conformation, notice that the steric positions of all seven Lys and N-Ala residues are same for both close and open conformation (see Figure S3), connections through Lys22, Lys70 and/or Lys165 might prevent the ‘lid’ structure from opening during the interfacial activation.36 Also, as is illustrated in Figure 1E and assuming that the BCL molecule is a perfect rigid sphere, covalently connection using Lys22, Lys70 and/or Lys165 might reduce the accessibility of the substrates. Instead, immobilization through Lys316 or N-Ala will allow the active center to be more accessible to the substrates. Therefore, we could infer that pNFD with higher activity would have higher portion of BCL connected through Lys316 and/or N-Ala. To the contrary, lower activity pNFD would have more of its BCL immobilized through Lys22, Lys70 and/or Lys165. However, connections through Lys70, Lys80, Lys269 and Lys283 are less likely to happen. 3.1.3 Mechanism of the immobilization reaction Interfacial activation plays an important role in the immobilization of BCL on pNFD (see Table S1 and Figure S4); the BCL molecule firstly approached the pNFD surface through interfacial activation and majority of them turned into open-conformation, and during this process, a covalent bond could be formed between the BCL molecule and pNFD. This whole process realized an immobilized open-conformation BCL molecule.


13


Page 14 of 39

Figure 2. FT-IR spectra of ST-DVB, FS-DVB, pFS-DVB, pNFD and pNFD-BCL Scheme 5. A proposed reaction routine Fuming nitric acid O 2N /H2SO4

NO2

Protein

O 2N

NO2

Weak base F

F

HN

Protein

Figure 2 compares the FT-IR spectra of ST-DVB (polymer of styrene and divinyl benzene), pFS-DVB, pNFD and pNFD-BCL. ST-DVB and FS-DVB had similar absorbance patterns but had several different points such as the absorbance peak at 1155 cm-1, which refers to the C-F bond (also present in the spectra of pNFD and pNFD-BCL). Therefore, both polymers have similar frameworks, but different side chain aromatic groups, indicating the successful integration of 4-fluorostyrene into the new polymer FS-DVB. After nitration, the double absorbance peak at 820-847 cm-1 indicated the stretching vibration of the two meta-position Hatoms on the aromatic ring, verifying a 1, 2, 3, 5-substitution. The peaks at 1349 cm-1 and 1533 cm-1 refers to the C-N vibration of the two –NO2 groups on the ring. This DNFB-like structure


14

Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


on pNFD showed similar reaction properties as that of DNFB because the two –NO2 groups on the ring strongly reduce the electronegativity of the C-F bond and, in turn, facilitates the nucleophilic substitution of ε-NH2 on the protein. Based on the above analysis, the actual synthesis routine is proposed in Scheme 5. Therefore, it can be safely concluded that the DNFBlike immobilization carrier was successfully synthesized. In the spectra of NFD-BCL, the weak absorbance at 1022 cm-1 (not seen in NFD) indicated the ε–NH2-C (aromatic) bond formed between the –NH2 on lipase and the aromatic C on the carrier by successfully substituting the F on the aromatic ring. This result agreed well with the mechanism of Sanger’s reaction (Scheme 1). Above all, the protein molecules were successfully immobilized on NFD via the ε–NH2 on Lys or α-NH2 on the N-terminal Ala residue. 3.2 Physical structure of FS-DVB, NFD, pNFD and pNFD-BCL In the suspension polymerization process without porogen, a transparent sphere with diameter of approximately 300 µm was collected (FS-DVB). Figure 3a-f demonstrates the morphological change of FS-DVB during the nitration and immobilization process. It is obvious that the shallow pits on the surface of FS-DVB become more evenly distributed (on NFD) after being processed with fuming nitric acid. The BET results for FS-DVB, NFD and NFD-BCL showed that this generally smooth surface sphere only has a specific surface area < 0.1 m2·g-1.


15


Page 16 of 39

Figure 3. SEM images of (a-b) FS-DVB, (c-d) NFD and (e-f) NFD-BCL; (a’-b’) pFS-DVB, (c’d’) pNFD and (e’-f’) pNFD-BCL with the corresponding pore width distribution A porous FS-DVB carrier (pFS-DVB) was synthesized to improve its immobilization capacity. As shown in Figure 3a’-b’, the pFS-DVB is a rough-surface sphere with a non-regular pore distribution. The specific surface area was improved from 50.02 m2·g-1 to 47.53 m2·g-1 (pNFD) after nitration (note that the specific surface area mentioned as follow are all acquired by BJH method for pore width larger than 10 nm, see Table S2). Meanwhile, the pore width distribution also became wider according to the pore width distribution plots in Figure 3b’and 3d’. Theoretically, more room was provided for BCL molecules. After the immobilization process, the immobilized protein should be located in or on the mesopores because it appeared slightly decrease according to the comparison of Figure 3d’and 3f’ with corresponding pore width distribution illustration. This decrease was also shown by the specific surface area decrease from 47.53 m2·g-1 to 43.42 m2·g-1, as well as a specific surface area contribution decrease shown in Figure S5. The final pNFD-BCL particles resembled mini golf-like spheres (Figure 3e’).


16

Page 17 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3 Basic enzyme properties of pNFD-BCL

Figure 4. Enzyme assay results of free BCL, pNFD-BCL and chitosan-BCL: (A) Effect of temperature on activity, (B) Effect of pH on activity, (C) Thermal stability assessment under 65℃, (D) Kinetic study based on Michaelis-Menten equation Figure 4 compares the effect of temperature, pH and thermal treatment of free BCL, pNFDBCL (note that subsequent studies both employed pNFD-BCL with immobilization capacity of 1.2 mg·g-1 and activity of 13.08±0.55 U·g-1) and chitosan-BCL (BCL immobilized on chitosan beads via a two-step cross-linking method37, immobilization capacity of 0.48 mg·g-1 and activity of 3.24 ± 0.14 U·g-1) as well as their kinetic study results.


17


Page 18 of 39

The pNFD-BCL improved the optimal reaction temperature from 40℃ to 65℃, even higher than that of the chitosan-BCL of 55℃ (Figure 4A). When the reaction temperature was increased from 30℃ to 55℃, the activity of pNFD-BCL showed an improvement while the compared groups remained stable. This phenomenon could be attributed to the pore structure of pNFD. At low temperature, mass transfer in pNFD-BCL was limited, lipase immobilized deep inside pores could not react with the substrates efficiently. But for the chitosan-BCL, as the chitosan beads carrier do not have a pore structure, all the immobilized proteins are cross-linked at its surface so that the mass transfer on it was not much limited. However, when temperature was further increased from 60℃ to 75℃, activities of the compared groups started to reduce, while the activity of pNFD-BCL remained stable at higher level compared to its original. This might be attributed to the accelerated substrates dynamic motion that make the deep immobilized BCL more available. In addition, the hierarchical structure of pNFD-BCL indeed act as a heat guardian to prevent the deactivation of deep immobilized BCL at high temperature. This could be reassured by its high thermal stability at 65℃. At even higher temperature, the cross-linked BCL on chitosan is relatively stable because the heat-led conformation distortion was highly restricted by many cross-linked covalent bound formed during the immobilization. In pNFD-BCL, as its numbers of connection with pNFD was only restricted by the few available Lys residues, its conformation might hence suffer a serious distortion to deactivation. This situation could also be applied to the free one as no peripheral immobilization connection was formed. The BCL immobilization on pNFD also led to a significant change of its optimal reaction pH (Figure 4B), from basic (pH 8-9) to neutral (pH 7). This change allows BCL to adapt to a wider


18

Page 19 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pH range. Owing to the hydrophobic nature of the pNFD surface, the H-bonds in the BCL molecule (H-BCL as followed) that keep its structure stable is hardly influenced after the immobilization process. In a lower pH reaction environment, the negative effect of higher concentration H+ on H-BCL is well defended by the hydrophobic surface of pNFD.20 By comparison, in the chitosan-immobilized BCL, the hydrophilic nature of chitosan beads can hardly avoid H+ attack, leaving the cross-linked BCL in a similar environment as that of free BCL. The thermal stabilities (Figure 4C) of pNFD was also improved as its activity did not change much during the first 4 h of heat treatment at 65℃, while the activity of free BCL and chitosanBCL remained at only 20% and 60%, respectively, of their original values. However, the activity of pNFD-BCL began to drop at a similar rate as that of free BCL after 4 h of treatment, while the activity of chitosan-BCL was relatively stable. This phenomenon could also be explained using the same theory mentioned above: the few Lys-ε-NH2-pNFD covalent connections leave BCL more easily subjected to conformational distortions at long-term heat treatment than the lipase multi-cross-linked on chitosan via numerous –NH2 groups on chitosan and glutaraldehyde. The opening/closing of the ‘lid’ structure is of high importance in the catalysis reaction and requires high flexibility of the lipase molecule. In the kinetic study (Figure 4D), pNFD-BCL showed a similar pattern as that of free-lipase as its molecular flexibility might not be much limited by its few bond connection(s). The Vmax of BCL only reduced by half to 238 µmol·L1

·min-1 while its substrates affinity (1/Km) increased by one-fold after immobilization on pNFD.

Therefore, this immobilized BCL still preserved the majority of its original performance. By contrast, the Vmax of cross-linked BCL on chitosan dropped by more than 80% compared to that


19


Page 20 of 39

of the free BCL because the multi-bond on the cross-linking chitosan carrier limited the flexibility of the BCL molecule. 3.4 Reusability in oil hydrolysis

Figure 5. Reusability test of pNFD-BCL in oil hydrolysis (sample withdrawn every 24 h) Figure 5 shows the result of the reusability test of pNFD-BCL in 72 h-per-batch RSO hydrolysis reactions. In the first 3×72 h reactions, the oil hydrolysis activity of pNFD-BCL was maintained above 90% of its original value, and reaction equilibrium was achieved within 48h, which could be explained by the fact that the pNFD particles just floated at their interface instead of evenly dispersing in oil or water phase. This should alleviate the mass transfer between oil, water and the solid immobilized enzyme as well as facilitate product-enzyme separation. However, its activity showed a continuous drop in the 4th to 7th batches. The main reason behind this drop is likely that the hydrophobic surface of pNFD favors the accumulation of glycerides, including the triglyceride, diglyceride. When the pores were covered by oil molecules, enzymes inside the pore were no longer available for the substrates, leading to apparent activity decreasing. Oil molecules absorbed on the surface could be washed with organic solvents, such


20

Page 21 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


as ether or 2-propanol, which would not exert negative influences on the activity of BCL.38 Second, long-term 45℃ processing of enzyme at the presence of water might lead to activity lose. In this situation, H+ or H-bonds from water could gradually influence the H-BCL structure and led to a chronic conformational distortion of BCL molecules. Other reasons, such as noncovalent connect BCL leaching or mechanical damage of the carrier, are both possibilities that lead the activity reduction. 3.5 Reusability in (R,S)-1-phenylethanol resolution

Figure 6. Reusability test of pNFD-BCL in (R,S)-1-phenylethanol resolution with vinyl acetate Figure 6 illustrates the performance of pNFD-BCL in the batch reaction for the resolution of (R,S)-1-phenylethanol using vinyl acetate as acyl donor. In the first 10 batch reactions, no activity or enantioselectivity loss was detected. The continuous batch reaction was suspended, and the recycled pNFD-BCL was stored at 4℃ for 8 weeks before the next 10 batches reaction. No apparent activity or enantioselectivity loss was detected before the 18th batch, in which the activity remained stable, while the enantioselectivity began to decrease. It was obvious that the pNFD-BCL was highly active and stable in the first 20 batch reactions even with an 8-week 4℃


21


Page 22 of 39

storage in between. The porous structure and hydrophobic nature of pNFD provides a stable microenvironment for BCL catalyzed non-aqueous reactions. Because the substrates are small molecules and of low viscosity, their accumulation did not cause the apparent activity loss as the triglycerides did. In this situation, the activity of BCL should be largely preserved in continuous batches of reactions. To test its limitation, the recycled pNFD-BCL was placed in open air at room temperature for one week before the next attempt. However, its activity and enantioselectivity both dropped sharply at the 21st run. Since the substrate (vinyl acetate) and co-solvent (n-heptane) used in this reaction are volatile at room temperature, their volatilization could simultaneously take away the very minimum structural water that intrinsically exists in BCL molecule to stabilize protein structure,39 and this process is irreversible. Nevertheless, the hydrophobic structure of pNFD is poor in moisture preserving. Therefore, as with the loss of structural water, the immobilized protein began to denature, resulting in a significant decrease of its activity and enantioselectivity. This loss could be avoided by storing pNFD-BCL in the reaction media in a closed lowtemperature environment. 4 Conclusion As inspired by the Sanger’s reagent, a new styrene polymer with the same mechanism was successfully synthesized and used as an enzyme immobilization support. This pNFD support performed well in the BCL (Burkholderia cepacia lipase) immobilization with an immobilization efficiency of 1.2 mg·g-1. However, due to the uncertainly of the randomly connected residue(s), the activity of the synthesized immobilized BCL showed a wide variance from 8.44 U·g-1 to 33.21 U·g-1, indicating a connection sites preference for better activity of the immobilized BCL. Based on steric analysis of the three-dimensional molecular surface of open-conformation BCL,


22

Page 23 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


we presumed that the immobilized connection via Lys316 and/or N-Ala would preserve a higher activity for BCL than via other Lys residues. Due to the hierarchical pore structure of pNFD, the optimal reaction temperature and thermal stability of the immobilized BCL were both enhanced compared to its free state. With the hydrophobic surface of pNFD, the pNFD-BCL showed a wider pH adaptation compared to its free counterpart. Substrate affinity was also improved according to its kinetic study. In the reusability test in different reaction environments, we found that the activity of immobilized enzyme was not only influenced by the substrates, but also by the interaction between substrates and surface microenvironment provided by the immobilizing carrier. Although the as synthesized pNFD-BCL showed good reusability for triglycerides hydrolysis and (R,S)-1-phenylethanol resolution, it still reserved the room of improvement. In summary, pNFD resin is a promising new material for enzyme immobilization because of its high performance in enzyme stabilization especially in non-aqueous reactions or in relatively high reaction temperature environment. To improve its performance in aqueous environment, surface graft would be needed to modulate the balance of hydrophobicity/hydrophilicity of the resin by making full use of the substitution sites on the aromatic ring in styrene polymer. In addition, the currently limited immobilization capacity of the support need improvement by optimizing pore structure and size of the resin spheres. ASSOCIATED CONTENT Supporting Information Supporting Information available: NMR analysis of product of Sanger’s reaction between threonine and DNFB at pH 8.0; preparing method of BCL solution for immobilization; procedure of pNpp hydrolysis reaction for enzyme assay; estimation of the BCL molecules distribution on


23


Page 24 of 39

pNFD carrier; analysis of the close conformation of BCL molecule; effect of Triton X-100 on the immobilization of BCL on pNFD; interpretation of the specific surface area available for immobilization; apparent activity data of the enzyme assay results. AUTHOR INFORMATION Corresponding Author *

Zhiyuan Wang, Tel.: +86 20 87065195, E-mail address: [email protected];

**

Pengmei Lv, Tel.: +86 20 87057727, E-mail address: [email protected].

ORCID Pengmei Lv: 0000-0001-9449-8652 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The financial support received from National Natural Science Foundation of China (No. 21576260), Special Funds of Applied Science and Technology Research of Guangdong Province (No.2015B020241002), Science and Technology Planning Project of Guangdong Province (No.2016A010104008) and Natural Science Foundation of Guangdong Province (No. 2015A030313720 and No.2017A010104010) is much appreciated. REFERENCES (1) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Cheml Soc Rev 2013, 42, 6223-6235. (2) Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; FernandezLafuente, R. Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties. Biomacromolecules 2013, 14, 2433-2462. (3) Shuttleworth, P. S.; De Bruyn, M.; Parker, H. L.; Hunt, A. J.; Budarin, V. L.; Matharu, A. S.;


24

Page 25 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Clark, J. H. Applications of Nanoparticles in Biomass Conversion to Chemicals and Fuels. Green Chem 2014, 16, 573-584. (4) Guzik, U.; Hupert-Kocurek, K.; Wojcieszynska, D. Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases. Molecules 2014, 19, 8995-9018. (5) Betancor, L.; Luckarift, H. R. Co-Immobilized Coupled Enzyme Systems in Biotechnology. Biotechnol Genet Eng 2010, 27, 95-114. (6) Min, K.; Yoo, Y. J. Recent Progress in Nanobiocatalysis for Enzyme Immobilization and its Application. Biotechnol. Bioprocess Eng 2014, 19, 553-567. (7) Hoffmann, C.; Chiaula, V.; Yu, L.; Pinelo, M.; Woodley, J. M.; Daugaard, A. E. Simple Preparation of Thiol-Ene Particles in Glycerol and Surface Functionalization by Thiol-Ene Chemistry (TEC) and Surface Chain Transfer Free Radical Polymerization (SCT-FRP). Macromolecular rapid communications 2018, 39, 1700394(1-6). (8) dos Santos, J. C. S.; Rueda, N.; Barbosa, O.; Fernandez-Sanchez, J. F.; Medina-Castillo, A. L.; Ramon-Marquez, T.; Arias-Martos, M. C.; Millan-Linares, M. C.; Pedroche, J.; Yust, M. D.; Goncalves, L. R. B.; Fernandez-Lafuente, R. Characterization of Supports Activated With Divinyl Sulfone as a Tool to Immobilize and Stabilize Enzymes via Multipoint Covalent Attachment. Application to chymotrypsin. Rsc Adv 2015, 5, 20639-20649. (9) Cao, L. Carrier-bound Immobilized Enzymes: Principles, Applications and Design, WileyVCH Verlag GmbH and Co. KGaA: Weinheim, Germany, 2005. (10) Abahazi, E.; Lestal, D.; Boros, Z.; Poppe, L. Tailoring the Spacer Arm for Covalent Immobilization of Candida antarctica Lipase B-Thermal Stabilization by Bisepoxide-Activated Aminoalkyl Resins in Continuous-Flow Reactors. Molecules 2016, 21, 767. (11) Hou, M.; Wang, R.; Wu, X. L.; Zhang, Y. F.; Ge, J.; Liu, Z. Synthesis of Lutein Esters by Using a Reusable Lipase-Pluronic Conjugate as the Catalyst. Catal Lett 2015, 145, 1825-1829. (12) Jia, F.; Narasimhan, B.; Mallapragada, S. Materials-Based Strategies for Multi-Enzyme Immobilization and Co-Localization: A Review. Biotechnol Bioeng 2014, 111, 209-222. (13) Sanger, F. The Free Amino Groups of Insulin. Biochem J 1945, 39, 507-515. (14) Heikens, D.; Hermans, P. H.; Vanvelden, P. F. Polymerization Reactions Occurring during Dinitrophenylation of Alpha-amino-acid, Beta-amino-acid and Omega-amino-acid with Sanger Reagent. Nature 1954, 174, 1187-1188. (15) Sanchez, D. A.; Tonetto, G. M.; Ferreira, M. L. Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions. Biotechnol Bioeng 2018, 115, 6-24. (16) Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; Bjorkling, F.; Hugejensen, B.; Patkar, S. A.; Thim, L. A Model for Interfacial Activation in Lipases from the Structure of a Fungal Lipase-Inhibitor Complex. Nature 1991, 351, 491-494. (17) Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernandez-Lafuente, R.; Huguet, J.; Guisan, J. M. Single Step Purification, Immobilization, and Hyperactivation of Lipases via Interfacial Adsorption on Strongly Hydrophobic Supports. Biotechnol Bioeng 1998, 58, 486-493. (18) Manoel, E. A.; dos Santos, J. C. S.; Freire, D. M. G.; Rueda, N.; Fernandez-Lafuente, R. Immobilization of Lipases on Hydrophobic Supports Involves the Open Form of the Enzyme. Enzyme Microb Tech 2015, 71, 53-57. (19) Baron, A. M.; Barouh, N.; Barea, B.; Villeneuve, P.; Mitchell, D. A.; Krieger, N. Transesterification of Castor Oil in a Solvent-free Medium Using the Lipase from Burkholderia cepacia LTEB11 Immobilized on a Hydrophobic Support. Fuel 2014, 117, 458-462. (20) Jin, Q. R.; Jia, G. Q.; Zhang, Y. M.; Yang, Q. H.; Li, C. Hydrophobic Surface Induced


25


Page 26 of 39

Activation of Pseudomonas cepacia Lipase Immobilized into Mesoporous Silica. Langmuir 2011, 27, 12016-12024. (21) Abahazi, E.; Boros, Z.; Poppe, L. Additives Enhancing the Catalytic Properties of Lipase from Burkholderia cepacia Immobilized on Mixed-Function-Grafted Mesoporous Silica Gel. Molecules 2014, 19, 9818-9837. (22) Fujiwara, M.; Shiokawa, K.; Yotsuya, K.; Matsumoto, K. Immobilization of Lipase from Burkholderia cepacia into Calcium Carbonate Microcapsule and its Use for Enzymatic Reactions in Organic and Aqueous Media. J Mol Catal B-Enzym 2014, 109, 94-100. (23) Hara, P.; Hanefeld, U.; Kanerv, L. T. Immobilised Burkholderia cepacia Lipase in Dry Organic Solvents and Ionic Liquids: A Comparison. Green Chem 2009, 11, 250-256. (24) Iyer, P. V.; Ananthanarayan, L. Enzyme Stability and Stabilization - Aqueous and Nonaqueous Environment. Process Biochem 2008, 43, 1019-1032. (25) Adlercreutz, P. Immobilisation and application of lipases in organic media. Cheml Soc Rev 2013, 42, 6406-6436. (26) Ansari, S. A.; Husain, Q. Potential Applications of Enzymes Immobilized on/in Nano Materials: A Review. Biotechnol Adv 2012, 30, 512-523. (27) DiCosimo, R.; McAuliffe, J.; Poulose, A. J.; Bohlmann, G. Industrial Use of Immobilized Enzymes. Cheml Soc Rev 2013, 42, 6437-6474. (28) Devendran, S.; Yadav, G. D. Lipase-Catalyzed Kinetic Resolution of (+/-)-1-(2-Furyl) Ethanol in Nonaqueous Media. Chirality 2014, 26, 286-292. (29) Rehman, S.; Wang, P.; Bhatti, H. N.; Bilal, M.; Asgher, M. Improved Catalytic Properties of Penicillium notatum Lipase Immobilized in Nanoscale Silicone Polymeric Films. International Journal of Biological Macromolecules 2017, 97, 279-286. (30) Li, X.; Liu, T.; Xu, L.; Gui, X. H.; Su, F.; Yan, Y. J. Resolution of Racemic Ketoprofen in Organic Solvents by Lipase from Burkholderia cepacia G63. Biotechnol. Bioprocess Eng. 2012, 17, 1147-1155. (31) Verma, M. L.; Puri, M.; Barrow, C. J. Recent Trends in Nanomaterials Immobilised Enzymes for Biofuel Production. Critical Reviews in Biotechnology 2016, 36, 108-119. (32) Verma, M. L.; Barrow, C. J.; Puri, M. Nanobiotechnology as a Novel Paradigm For Enzyme Immobilisation and Stabilisation with Potential Applications in Biodiesel Production. Applied Microbiology and Biotechnology 2013, 97, 23-39. (33) Fu, J. Y.; Chen, L. G.; Lv, P. M.; Yang, L. M.; Yuan, Z. H. Free Fatty Acids Esterification for Biodiesel Production Using Self-synthesized Macroporous Cation Exchange Resin as Solid Acid Catalyst. Fuel 2015, 154, 1-8. (34) Wang, J. Y.; Ma, C. L.; Bao, Y. M.; Xu, P. S. Lipase Entrapment in Protamine-induced BioZirconia Particles: Characterization and Application to the Resolution Of (R,S)-1-Phenylethanol. Enzyme Microb Tech 2012, 51, 40-46. (35) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of Enzyme Activity, Stability and Selectivity via Immobilization Techniques. Enzyme Microb Tech 2007, 40, 1451-1463. (36) Barbe, S.; Lafaquiere, V.; Guieysse, D.; Monsan, P.; Remaud-Simeon, M.; Andre, I. Insights into Lid Movements of Burkholderia cepacia Lipase Inferred from Molecular Dynamics Simulations. Proteins 2009, 77, 509-523. (37) Hung, T. C.; Giridhar, R.; Chiou, S. H.; Wu, W. T. Binary Immobilization of Candida rugosa Lipase on Chitosan. J Mol Catal B-Enzym 2003, 26, 69-78. (38) Liu, Y.; Zhang, X.; Tan, H.; Yan, Y.; Hameed, B. H. Effect of Pretreatment by Different


26

Page 27 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Organic Solvents on Esterification Activity and Conformation of Immobilized Pseudomonas cepacia Lipase. Process Biochem 2010, 45, 1176-1180. (39) Li, C.; Tan, T.; Zhang, H.; Feng, W. Analysis of the Conformational Stability and Activity of Candida antarctica Lipase B in Organic Solvents Insight from Molecular Dynamics and Quantum Mechanics/Simulations. J Biol Chem 2010, 285, 28434-28441.

Table of Contents Graphic


27


Figure 1. Preferred amino acid residue connection sites analysis based on the steric molecular structure of BCL 279x185mm (150 x 150 DPI)


Page 28 of 39

Page 29 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. FT-IR spectra of ST-DVB, FS-DVB, pFS-DVB, pNFD and pNFD-BCL 71x76mm (600 x 600 DPI)



Figure 3. SEM images of (a-b)FS-DVB, (c-d)NFD and (e-f)NFD-BCL; (a’-b’)pFS-DVB, (c’-d’)pNFD and (e’f’)pNFD-BCL with the corresponding pore width distribution 619x285mm (150 x 150 DPI)


Page 30 of 39

Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Enzyme assay results of free BCL, pNFD-BCL and chitosan-BCL: (A) Effect of temperature on activity, (B) Effect of pH on activity, (C) Thermal stability assessment under 65℃, (D) Kinetic study based on Michaelis-Menten equation 894x708mm (96 x 96 DPI)



Figure 5. Reusability test of pNFD-BCL in oil hydrolysis (sample withdrawn every 24 h) 85x41mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Reusability test of pNFD-BCL in (R, S)-1-phenylethanol resolution with vinyl acetate 74x33mm (300 x 300 DPI)



Table of Content Graphic 308x160mm (150 x 150 DPI)


Page 34 of 39

Page 35 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure S1. NMR 1H spectrum of the product of DNFB and Thr at pH 8.0 (50mM, PBS), 37℃, 24h 297x209mm (200 x 200 DPI)



Figure S2. NMR 13C spectrum of the product of DNFB and Thr at pH 8.0 (50mM, PBS), 37℃, 24h 297x209mm (200 x 200 DPI)


Page 36 of 39

Page 37 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure S3. Modulated 2D structure of BCL molecular (Adapted from reference1) 384x237mm (150 x 150 DPI)



Figure S4. Interfacial activation effect of Triton X-100 on the covalent immobilization of BCL on pNFD 346x208mm (150 x 150 DPI)


Page 38 of 39

Page 39 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure S5. Specific surface area contribution by pore width (data acquired by BJH method) 67x57mm (300 x 300 DPI)


Novel Sanger's Reagent-like Styrene Polymer for the Immobilization of

Recommend Documents