Specific Ligation of Two Multimeric Enzymes with Native Peptides and

Subscriber access provided by Fudan University

Article

Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio Kun Du, Jinjin Zhao, Jian Sun, and Wei Feng Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00043 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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 free 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 accessible to all readers and 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.

Bioconjugate Chemistry 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 31

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

Bioconjugate Chemistry

Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio Kun Du†, Jinjin Zhao†, Jian Sun, Wei Feng Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, 100029, China

ABSTRACT: D-amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H2O2 during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in a close proximity to the CAT subunits, facilitating immediate transfer of H2O2 from one catalytic site to the other, enabling efficiently decomposing the generated H2O2. The reduced cofactors of the DAAO subunits were re-oxidized by the evolved molecular oxygen around. Kinetics analysis showed that the D-alanine substrate follows Michaelis–Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4 fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3±2.7%. The encapsulated DAAO&CAT has retained 98.1±3.1% and 94.9±2.9% of the activity of free DAAO&CAT at 30 °C and 40 °C, respectively. 1

ACS Paragon Plus Environment


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 31

INTRODUCTION Various reactions for manufactures of pharmaceuticals, foods, and chemicals have been catalyzed by enzymes.1 Some reactions are catalyzed by multi-enzymes in a cascade mode.2 The cooperation of enzymes facilitates the transfer of reaction intermediates between the active sites of different enzymes, thus reactivity and selectivity can be enhanced. Many enzymes in biotechnological applications are multimeric proteins: oxidases,3 catalases,4 dehydrogenases,5 galactosidases,6 etc. D-amino acid oxidases (DAAOs) are flavoenzymes that catalyze a D-amino acid substrate into α-keto acid, in the meantime H2O2 is evolved. DAAOs have been used for oxidation of cephalosporin C,8 production of α-keto acids,9 catalyzing the deamination of aromatic, aliphatic, and polar amino acids,10 and deracemization of racemic amino acids for the synthesis of pharmaceuticals.11 Together with transaminases,

DAAOs

have

been

used

for

producing

precursors

for

pharmaceuticals.12 However, hydrogen peroxide is generated when DAAOs catalyze the reactions. Hydrogen peroxide can partially destroy DAAOs,13 and accumulation of evolved hydrogen peroxide can cause by-product inhibitory effect.14 These two aspects lead to the reduction of the catalytic efficiency of DAAO.15 To overcome the negative effect of hydrogen peroxide, catalase (CAT) and horseradish peroxidase (HRP) have been used with DAAOs in order to decompose the hydrogen peroxide of DAAO.16-18 To construct such two-enzyme systems, for example, co-immobilization of CAT and DAAO has been investigated.19,20 However, through co-immobilization, it is impossible to spatially arrange CAT or HRP in proximity to DAAO with a precision control of enzyme molar ratio. Aggregation of two enzymes through cross-linking could not control the enzyme molar ratio either, and only 40% of enzymatic activity was recovered.21 There are successful examples for linking two 2


Page 3 of 31

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


enzymes by gene fusion.22 However, for DAAO and CAT, our results demonstrated that when the genes of DAAO and CAT were fused and expressed in E. coli, the fusion protein did not exhibit any enzymatic activity. It is possibly ascribed to that the enzymes after fusion could not be folded correctly, as both enzymes are multimeric proteins and have large molecular weights. In addition, for multimeric enzymes, the subunit–subunit interactions may be weakened under certain experimental conditions.23 DAAO and CAT also face the same concern. The enzymes with a moderate-to-low stability are not favored for industrial applications. This work intends to establish a two-enzyme system by specifically linking DAAO with CAT, in order to improve the enzymatic activity and stability of DAAO. Protein splicing is accomplished through self-excision of inteins and concomitant ligation of exteins.24,25 Supplementary Scheme S1 schematically shows split intein-mediated protein trans-splicing. The N- and C-terminal parts of the split intein fold into the active intein domain, and then exteins are ligated with a peptide bond.26,27 Protein splicing has been used for cyclization of peptides and proteins,28-30 segmental isotope labeling for protein NMR,31 and intein-triggered artificial protein hydrogels.32 Herein through intein-mediated protein splicing, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides. The spliced product DAAO&CAT has been further encapsulated within a coordination polymer. The catalytic efficiency of the spliced product DAAO&CAT has been evaluated by catalyzing the D-alanine deamination. The results of this work have implications for establishing other two multimeric enzyme systems.

RESULTS AND DISCUSSION Enzyme expression and in vivo splicing. Protein splicing is achieved through self-excision of inteins and concomitant ligation of exteins (Scheme S1).33 DAAO 3



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 4 of 31

contains two subunits, each subunit has a molecular weight 43 kDa. CAT has four subunits, each subunit has a MW 57.3 kDa. The C-terminal part of the split NpuDnaE intein34 (IntC) is 36 amino acids in length, which is fused to the N-terminus of the DAAO subunit, and the N-terminal part of the split NpuDnaE intein (IntN) is 102 amino acids in length, which is fused to the C-terminus of the CAT subunit. For the ligation of the two multimeric enzymes, it has been demonstrated that linkers between IntC and the DAAO subunit and between IntN and the CAT subunit are required (Figure S1). Without the linkers, the spliced product DAAO&CAT cannot be observed through the SDS-PAGE analysis. Both flexible and helical linkers have been used. However, with a flexible linker, such as GGGGSGGGGSGGGGS, there is no spliced product. While with a rigid linker, EPPPPLPPPPLPPPPEPPPP, the spliced product DAAO&CAT is obtained. It can be reasoned that the steric hindrance for the splicing has been significantly reduced by using the rigid linker, revealing a subtle role of the rigid linker in the multimeric enzyme splicing. The spliced product DAAO&CAT was purified through three procedures, primary purification by Ni Sepharose beads, further fractionation by ultrafiltration (50 KDa cut off), and final purification by gel filtration chromatography. Such purification ensures that those enzymes DAAO and CAT that did not take part in the splicing reactions have been separated from DAAO&CAT. The purified spliced product DAAO&CAT was first subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Due to the protein denaturation by SDS, the subunits, including DAAO-CAT, CAT-link-IntN, CAT-link, and IntC-link-DAAO, appeare as separate bands (Figure 1a). Appearance of the bands for IntC-link-DAAO and CAT-link-IntN subunits is ascribed to that, these subunits did not taken part in the splicing, but they are associated with the DAAO-CAT subunit (Figure 1c). In addition, 4


Page 5 of 31

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


during the splicing, some N-terminal (IntN) parts are cleaved off from CAT-link-IntN.35 As a result, the band for the CAT-link subunit appears. Appearance of the band of the DAAO-CAT subunit confirms that the ligation of the two enzymes indeed occurs through the in vivo splicing. The DAAO-CAT band appears just below the position marked by the Mark protein (116 kDa), in agreement with the theoretically predicted molecular weight of 100.3 kDa. We have used MALDI-TOF-MS to determine the molecular weights (MWs) of the DAAO&CAT fractions. However, reliable data could not be obtained, possibly due to that the DAAO&CAT fractions have large molecular weights. To investigate the distribution of the DAAO&CAT fractions and estimate their molecular weights, the spliced product DAAO&CAT was subjected to blue native polyacrylamide gel electrophoresis (BN-PAGE), which has been used to identify protein fractions of multiprotein complexes.36 In Figure 1b for the BN-PAGE, two broadened bands for DAAO&CAT appears. Figure S2 shows the chromatogram for the DAAO&CAT fractions analyzed by the AKTAFPLC system. The two peaks are for DAAO&CAT I and DAAO&CAT II, respectively. The chromatogram is consistent with the BN-PAGE (Figure 1b), which shows two bands representing DAAO&CAT I and DAAO&CAT II. The apparent molecular weights (AMWs) of the DAAO&CAT fractions were estimated by comparing their electrophoretic mobility with that of Mark proteins (supplementary Figure S3). The band DAAO&CAT I covers AMWs from 601k to 550k. Considering the MWs of CAT-link-IntN and IntC-link-DAAO, two linking modes can satisfy this AMW range. They are CAT−DAAO−CAT and CAT−4DAAO (Table S2). The linking mode CAT−4DAAO means that each of the four CAT-link subunits has been ligated to a link-DAAO subunit. However, such splicing reaction will encounter significant steric hindrance, as both the CAT-link-IntN 5



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 6 of 31

and IntC-link-DAAO subunits have large molecular weights (Table S2). Hence we can exclude the linking mode of CAT−4DAAO for the in vivo splicing. The band DAAO&CAT II covers AMWs from 389k to 310k. Only the linking mode DAAO-CAT can satisfy this AMW range (Table S2). This broadened band is possibly ascribed to that some N-terminal (IntN) part was cleaved off from the CAT-link-IntN subunit during the splicing reaction. The BN-PAGE analysis (Figure 1b) revealed that the DAAO&CAT fractions results from the two linking modes CAT−DAAO−CAT and CAT−DAAO (Figure 1c). These two linking modes imply that the not-linked IntC-link-DAAO subunit has a smaller molar fraction compared to that of not-linked CAT-link-IntN and CAT-link subunits. This is consistent with the SDS-PAGE in Figure 1a, in which the band of not-linked IntC-link-DAAO subunit appears with a relatively

weak

intensity.

The

middles

of

the

bands

DAAO&CAT

I

(CAT−DAAO−CAT) and DAAO&CAT II (DAAO−CAT) migrate at the positions 567.28±3.79k and 333.84±2.02k, respectively. Based on the difference in intensity of the bands, the relative abundances are 78.2% for CAT−DAAO−CAT and 21.8% for DAAO−CAT. The apparent molecular weight of DAAO&CAT has been estimated to be 516.39k.

6


Page 7 of 31

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


1

M

kDa 200

DAAO-CAT subunit

1

2

3

M

116 97.2

kDa 1236 1048

N

CAT-link-Int subunit CAT-link subunit IntC-link-DAAO subunit

66.4

DAAO&CAT I (CAT−DAAO−CAT)

44.3

DAAO&CAT II (DAAO−CAT)

720 480 242 146 66

29

b (BN-PAGE)

a (SDS-PAGE)

IntC -link-DAAO

DAAO-CAT

DAAO-CAT

CAT -link-Int DAAO

CAT -DAAO-CAT

N

CAT

IntN

Intein

CAT-DAAO-CAT

CAT -link-IntN

DAAO

CAT

Int

IntN

IntC

Linker

c (DAAO&CAT fractions) Figure 1. (a) Analysis of the spliced product DAAO&CAT by SDS-PAGE. Lane M: marker; lane 1: sample. (b) Analysis of the spliced product DAAO&CAT by BN-PAGE. Lane M: marker; lanes 1, 2, and 3 for samples 1, 2, and 3, respectively. (c) Schematic diagram representing the DAAO&CAT fractions resulted from the linking modes of CAT-DAAO-CAT and DAAO-CAT. The two precursors IntC-link-DAAO and CAT-link-IntN were co-expressed in E. coli, and the in vivo splicing occurred after the expression of precursors. The link is 20 amino acids in length.

Stability of the spliced product DAAO&CAT. Activity assay has demonstrated that the spliced product DAAO&CAT can catalyze D-alanine deamination and decomposition of H2O2, meaning that the activities of DAAO and CAT have been retained by DAAO&CAT. It is implied that the original quaternary structures of the enzymes have been preserved after the in vivo splicing. To investigate the change of 7


产物


the secondary structures after splicing, circular dichroism (CD) spectra have been measured for the spliced product DAAO&CAT and the mixed enzymes DAAO+CAT. For each case, equal amount of proteins with the same molar ratio of DAAO to CAT was used. Figure 2 shows that the two CD spectra are close to each other. It is indicative of that the in vivo splicing has little effect on the secondary structures of the enzymes.

6 4

CD [mdeg]

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 8 of 31

DAAO+CAT DAAO&CAT

2 0 -2 -4

200

210

220

230

240

250

260

Wavelength (nm)

Figure 2. CD spectra for the spliced product DAAO&CAT and mixed enzymes DAAO+CAT

Fluorescence spectroscopic experiments were performed to investigate the stability of DAAO and DAAO&CAT using urea as denaturing agent. Tryptophan is the dominant fluorescent amino acid. 37 Herein, the intrinsic fluorescence of the enzymes was quenched by acrylamide, in order to investigate protein conformation change upon interfering by the denaturing agent. The fluorescence spectra with excitation of 295 nm are shown in Figures 3a and 3b. The Stern-Volmer equation F0/F=1+ KSV[Q] can be used to describe the concentration-dependence quenching,37 where F and F0 are the fluorescence intensities in the presence and absence of acrylamide, respectively, [Q] is the acrylamide concentration, and KSV is the Stern-Volmer quenching constant. By

8


Page 9 of 31

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


comparing KSV values, the information of conformational change can be obtained.37 A larger KSV value means a lower stability. Based on the fluorescence spectra (Figures 3a and 3b), the Stern-Volmer plots for DAAO and DAAO&CAT were obtained (Figure 3c). The KSV value for DAAO at the urea concentration 6 M is 19.85, in comparison to 13.03 for DAAO&CAT. Compared to DAAO&CAT, DAAO is more inclined to exposure of its tryptophan residues to solvent upon interfering by urea. DAAO&CAT is more resistant to the urea denaturation than DAAO. The spliced product DAAO&CAT is a result by linking the subunit of IntC-link-DAAO with the subunit of CAT-link-IntN, leading to a lower mobility of the subunits involved in subunit-subunit multi-interactions. DAAO&CAT may become more rigid than DAAO. The results suggest that linking subunits of different enzymes with native peptides through in vivo splicing is an approach to enhancing the stability of multimeric enzymes.

Figure 3. (a, b) Change of fluorescence intensity of the proteins with acrylamide concentration (M); (c) Stern-Volmer plots for the quenching of protein fluorescence with acrylamide. F0 and F are the fluorescence intensities in absence and presence of acrylamide, respectively. 9



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 10 of 31

Multi-enzyme catalysis. DAAO&CAT combines the activities of DAAO and CAT. DAAO&CAT has been first tested for its ability to decompose H2O2. In the presence of H2O2, the oxidation of 3,5,3'5'-tetramethylbenzidine (TMB) was catalyzed by DAAO&CAT. Before addition of H2O2, three systems as reference were tested as shown in Figure S4. When H2O2 was added to the TMB solution and incubated for 10 min, the change of solution colour was not observed (Figure 4), indicating that TMB was not oxidized during the period of time. When TMB and H2O2 were incubated with DAAO&CAT for 10 min, H2O2 was decomposed by DAAO&CAT, TMB was oxidized concomitantly, and the solution exhibited a blue colour (Figure 4).38 Four additional systems have been tested as well, including H2O2+TMB, MnO2+TMB, H2O2+TMB+DAAO+CAT, H2O2 +TMB+DAAO&CAT. Figure S5 shows that after 10 min incubation, the color of the solution of H2O2 +TMB has little change, and the absorbance at 369 and 650 nm are very small (black line in Figure S5b). For another two systems, H2O2+TMB+DAAO+CAT and H2O2+TMB+DAAO&CAT, the same amount of enzyme was used. The color change of the solutions and the UV-vis absorbance demonstrated that with the same amount of enzyme, DAAO&CAT is advantage over the mixed enzymes DAAO+CAT.

DAAO

DAAO&CAT

Figure 4. Decomposition of hydrogen peroxide by DAAO&CAT Systems: (a) 100 mM H2O2 + 2 mM TMB + 0.1 mg/mL DAAO; (b) 100 mM H2O2 + 2 mM TMB+ 0.1 mg/mL DAAO&CAT. All the reactions were carried out in a PBS buffer (pH 5.0) at 30℃ for 10 min. 10


Page 11 of 31

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


In the reaction of D-alanine deamination, D-alanine is converted to pyruvate, and H2O2 is generated. Here the reactions were catalyzed by DAAO and DAAO&CAT. Qualitative comparison of enzymatic activity was carried out by addition of 2,4-dinitrophenylhydrazine (DNPH), which can react with generated pyruvate to form a colored product.39 The solution with DAAO exhibits a colour in yellow (Figure 5a), while the solution with DAAO&CAT exhibits a brownish-red color (Figure 5a). It is indicated that during the same period of reaction time, more D-alanine has been converted to pyruvate under the catalysis of DAAO&CAT. For the D-alanine deamination reaction, the spliced product DAAO&CAT has also been compared with the mixed enzymes DAAO+CAT. The tube containing DAAO&CAT exhibits a deeper color than the tube containing DAAO+CAT (Figure S6a). The colored product exhibits characteristic peaks at 438 nm. Figure S6b shows that the solution containing DAAO&CAT has a larger absorbance than the solution containing DAAO+CAT. The results in Figure S6 demonstrate that DAAO&CAT exhibits a higher catalysis efficiency than the mixed enzymes DAAO+CAT. The result of the qualitative comparison has demonstrated that DAAO&CAT exhibits a higher activity than DAAO (Figure 5a). To have a quantitative comparison, enzyme kinetics has been investigated. Figure 5b shows plots of the initial rate of reaction (V0) as a function of the substrate concentration [S] that obey the Michaelis-Menten Kinetics. At each substrate concentration, the initial rates of reactions by DAAO&CAT are larger than that by DAAO, meaning a higher substrate consuming rate by DAAO&CAT. The data were regressed using the function expressed for the Michaelis-Menten kinetics to obtain the kinetics parameters (Table 1). DAAO&CAT exhibits a Km value much smaller than that of DAAO, indicating that the spliced product DAAO&CAT has significantly improved the affinity towards 11



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 12 of 31

the substrate.40 The Kcat/Km ratio has been used to measure catalysis efficiency.40 The Kcat/Km ratio of DAAO&CAT is 22.4 times that of DAAO, indicating a much higher catalytic efficiency achieved by DAAO&CAT. This kinetics result is in accordance with the qualitative comparison as shown in Figure 5a. The analysis of enzymatic activity shows that the spliced product DAAO&CAT has significantly improved the activity of D-amino acid oxidase. Through the in vivo splicing, the DAAO subunits and CAT subunits are ligated, thus the two enzymes are spatially arranged in a highly controlled manner, facilitating the sequential catalysis. The spliced product DAAO&CAT coordinates the activities of DAAO and CAT. The by-product H2O2 of DAAO is the substrate of CAT. Molecular dynamics simulations demonstrated that the center-of-mass distance between DAAO and CAT of the DAAO-CAT subunit in DAAO&CAT is about 6.05 nm (Figure S7). Due to the proximity between the DAAO and CAT subunits, the transfer of H2O2 from the active sites of the DAAO subunits to the active sites of the CAT subunits is facilitated. Thus the CAT subunits immediately catalyze the decomposition of H2O2. Flavin adenine dinucleotide (FAD) is a cofactor of DAAOs. The oxygen evolved from the decomposition of H2O2 is used to oxidize the cofactor, it is reduced during the catalysis. Thus the hydrogen peroxide→oxygen→hydrogen peroxide cycles are formed as illustrated in Figure 5c. Formation of the cycles has several advantages: eliminating the deleterious and inhibitory effects by H2O2,41 and supplying molecular oxygen to oxidize the reduced FAD for maintaining its function. Thus the deamination reaction can be accelerated.

12


Page 13 of 31

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


a

c

DAAO-CAT subunit

DAAO-CAT CAT-DAAO-CAT

Figure 5. (a) Qualitative comparison between DAAO&CAT and DAAO for catalyzing the deamination of D-alanine. (b) Plots of the initial rate of reaction (V0) as a function of the substrate concentration [S] for the enzymes DAAO&CAT and DAAO. (c) Schematic presentation for the formation of hydrogen peroxide→oxygen→hydrogen peroxide cycles under the catalysis of DAAO&CAT. DAAO&CAT catalyzes the conversion of D-alanine to pyruvate, in the meantime 13



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 14 of 31

catalyzes the decomposition of the generated H2O2. The reduced cofactor FAD of DAAO is oxidized by the evolved oxygen. The deamination reaction is accelerated by the formation of the hydrogen peroxide→oxygen→ hydrogen peroxide cycles. Table 1. Kinetics parameters for DAAO and DAAO&CAT DAAO DAAO&CAT Vm (mM.min-1) 0.0287 0.0429 Km (mM) 15.37 6.37 Kcat (min-1) 1155.8 10747.2 Kcat/ Km (mM-1 min-1) 75.2 1687.1 The parameters were obtained based on Figure 5b. The enzyme concentrations for DAAO and DAAO&CAT are 0.002 mg/ml, respectively.

Encapsulation of the spliced product DAAO&CAT within a coordination polymer (CP). Co-immobilization of two enzymes onto/within a support is an approach to simultaneously utilize the two enzymes for sequential catalysis.16-18 However, it is very difficult/impossible to spatially arrange an enzyme in close proximity to another enzyme with a precision control of enzyme molar ratio. While for the spliced product DAAO&CAT, DAAO has been ligated to CAT with a peptide through the in vivo splicing. This facilitates the co-immobilization of the two enzymes in close proximity to each other with a precisely controlled molar ratio. Various materials have been investigated for immobilization of enzymes. Herein we used a coordination polymer (CP) to encapsulate the spliced product DAAO&CAT. Recently coordination polymers or metal-organic frameworks have been extensively investigated for encapsulation of enzymes by taking advantage of the protective properties.42-45 Standard ZIF-8 exhibits truncated cubic crystals (Figure 6a) with sizes less than 0.5 µm3. The powder X-ray diffraction (PXRD) analysis revealed that that the synthesized ZIF-8 exhibits crystals (Figure S8). After encapsulation of DAAO&CAT by the coordination polymer, the CP/DAAO&CAT composite exhibits spherical and oval shapes (Figure 6b), and the composite sizes are much larger than that of standard ZIF-8. The PXRD for CP/DAAO&CAT indicates that the composite 14


Page 15 of 31

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


exhibits mostly amorphous morphology. The morphology and size of the composite clearly demonstrated that the immobilization of DAAO&CAT was accomplished by encapsulation. Additional evidence of DAAO&CAT encapsulation was obtained by the image of confocal microscopy. The spliced product DAAO&CAT was tagged with fluorescein isothiocyanate (FITC) and encapsulated by the CP. The resultant composite was washed with Triton X-100 and subjected to confocal microscopy. Homogenously luminescent cross-sections of the CP/DAAO&CAT composite are observed (Figure 6c). A 77 K nitrogen adsorption isotherm was measured for the composite of CP/DAAO&CAT (Figure S9). BET analysis of the data indicates that CP/DAAO&CAT has a surface area of 1090 m2 g-1, showing a decrease of porosity relative to ZIF-8 with a surface area of 1603 m2 g-1. This is due to the presence of DAAO&CAT.

a (scale bar: 1.0 µm)

b (scale bar: 10 µm)

c Figure 6. (a) SEM image of standard ZIF-8; (b) SEM image of CP/DAAO&CAT ; (c) Confocal laser scanning microscopy image of CP/DAAO&CAT (labeled with FITC). 15



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 16 of 31

The CP/DAAO&CAT composite has been tested for its enzymatic activity at various temperatures (Figure 7). At 30 and 40 °C, the composite has retained 98.1±3.1% and 94.9±2.9% of the activity of free DAAO&CAT, respectively. At 50 °C, the composite exhibits a higher activity than free DAAO&CAT. Further elevating reaction temperature to 60 °C, the activity of free DAAO&CAT has reduced significantly. Free DAAO&CAT has completely lost the activity at 70 °C and 80 °C. In contrast, the composite has retained 72.1±3.2% at 60 °C, 69.7±2.5% at 70 °C, and 64.1±3.5% at 80 °C of its activity at 30 °C.

Figure 7. Enzymatic activity for free DAAO&CAT and CP/DAAO&CAT

CONCLUSIONS The N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits through in vivo protein splicing. The two enzymes are linked with peptide bonds, which are formed naturally within the expressing host. The secondary structures of the two enzymes have been preserved in the spliced product DAAO&CAT. The spliced product DAAO&CAT has exhibited a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are spatially arranged in a close proximity to the CAT subunits at a molecular distance, enabling efficient transfer of the intermediate between the active sites. As a result, the catalytic 16


Page 17 of 31

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


efficiency of DAAO&CAT is 22.4 fold that of DAAO. Furthermore, the spliced product DAAO&CAT have been encapsulated within a coordination polymer. Immobilization of DAAO&CAT means that the two enzymes have been immobilized simultaneously with precisely controlled molecular ratio. The encapsulated DAAO&CAT has retained a high percentage of the activity of free DAAO&CAT in a wide range of reaction temperatures. This work has presented an efficient methodology for establishing multi-enzyme systems by specifically linking two multimeric enzymes. The methodology overcomes the problems of protein folding encountered in the fusion of multimeric enzymes.

MATERIALS AND METHODS Restriction enzymes, DNA polymerase and DNA ligase were obtained from New England Biolabs and Fermentas. Oligonucleotide primers were synthesized by BGI Tech (Shenzhen, China). All other reagents were purchased from Sinopharm Chemical Reagent Co.,Ltd (Shanghai, China) or Sigma-Aldrich(Shanghai, China) and used without additional purification processes. Construction of vectors. Table S1 lists the primers required. “fp”and “rp” stand for forward and reverse primers, respectively. The genomic DNA of Trigonopsis variabilis was used as template, the gene of DAAO was amplified by using the paired primers DAAO-fp and DAAO-rp. After purification by using a DNA extraction kit (Omega Bio-tek), the DAAO gene was codigested with HindIII and BamHI restriction endonucleases. The resulting fragment was ligated into the plasmid pETDuet-1, which has been codigested with HindIII and BamHI restriction endonucleases. Thus the expression vector pETDuet/DAAO in Escherichia coli was constructed containing a C-terminal hexa histidine tag. The IntC-linker gene was amplified by PCR using the paired primers IntC-fp and IntC-rp, then purified and codigested with NcoI and BamHI 17



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 18 of 31

restriction endonucleases. The resulting fragment was ligated into the plasmid pETDuet/DAAO, which has been codigested with NcoI and BamHI restriction endonucleases.

Thus

the

expression

vector

pETDuet/IntC-link-DAAO

was

constructed. The catalase (CAT) gene was amplified by PCR with genomic DNA (Bacillus subtilis 168) using primers CAT-fp and CAT-rp (Table S1). The purified CAT gene was codigested with NdeI and KpnI restriction endonucleases. The resulting fragment was ligated into the plasmid pETDuet/IntC-link-DAAO, which has been codigested with NdeI and KpnI restriction endonucleases. Thus the expression vector pETDuet/IntC-link-DAAO/CAT in Escherichia coli was constructed. The IntN-linker gene was amplified by PCR using the paired primers IntN-fp and IntN-rp, then purified and codigested with KpnI and XhoI restriction endonucleases. The resulting fragment was ligated into the plasmid pETDuet/IntC-DAAO-CAT, which has been codigested with

KpnI

and

XhoI

restriction

endonucleases.

Finally

the

vector

pETDuet/IntC-link-DAAO/CAT-link-IntN was constructed as illustrated in Figure S1. The linkers between IntC and DAAO and between IntN and CAT are a 20 amino acid rigid linker domain EPPPPLPPPPLPPPPEPPPP. For expressing CAT, the vector pET28a/CAT was constructed containing a C-terminal hexa histidine tag. Protein expression and in vivo splicing. E. coli BL21 cells harboring various plasmids were grown at 37 °C in LB medium with 50 µg/ml Amp to an OD600 of 0.45, were induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and were grown for an additional 8 h at 25°C for expressing DAAO and CAT, or 25°C for coexpressing the precursors IntC-link-DAAO and CAT-link-IntN and the in vivo splicing. Prolonging the process time had no significant improvement in the production of the spliced product DAAO&CAT. The E. coli cells were harvested by 18


Page 19 of 31

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


centrifugation at 5000 g for 30 min at 4°C and resuspended in a buffer (20 mM Tris-Cl, pH8.0, 300 mM NaCl, 1% Triton). The cells were lysed by ultrasonic disruption in ice water, and the lysate was centrifuged at 10,000 g at 4 °C for 30 min to remove cell debris. The supernatant was transferred to a fresh tube. Protein purifications were first performed using Ni Sepharose beads (GE Life Sciences) in a chromatography column (GE Life Sciences) and eluted with 500 mM imidazole. The imidazole was removed from the purified proteins by dialyzing into a buffer (50 mM Tris-Cl pH 7.5, 500 mM NaCl, 1 mM EDTA). The purified proteins containing the spliced products DAAO&CAT were further fractionated by ultrafiltration (GE Healthcare, 50 KDa cut off), in order to remove the not-spliced precursor IntC-link-DAAO. The spliced product DAAO-CAT was finally purified by gel filtration chromatography on a Superose 6 column (AKTA Pure chromatography system, GE Healthcare), using an elution buffer (50 mM Phosphate, pH 7, 150 mM NaCl). Estimation of apparent molecular weight for the spliced products. Analysis of the spliced products by blue native PAGE was described in Supporting Information. High resolution gel images were obtained by scanning at 600 DPI. Adobe Photoshop (version 13) was used to process the gel images to obtain the best contrast for densitometry analysis. Relative abundances of the DAAO&CAT fractions were determined by optical densitometry using Gel-Pro Analyzer software program. The apparent molecular weights of the DAAO&CAT fractions were determined by comparing the electrophoretic mobility of the DAAO&CAT fractions with that of Mark proteins. The Mark proteins (Invitrogen) for native electrophoresis are in the range from 20 to 1236 kDa. The standard curve for the relative mobility of Mark proteins for native electrophoresis is shown in Figure S2. 19



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 20 of 31

Circular dichroism (CD) spectroscopy. CD spectra were measured on a JASCO J-810 CD instrument to monitor the change of secondary structures of the enzymes. The bandwidth was 0.5 nm and the scan speed was 50 nm/min. Cell length was 1 cm. The enzyme solutions were prepared by dissolving the enzymes in PBS buffer. The enzyme concentrations were 0.1 mg/mL, and the measurements were performed at 25 °C. The CD spectrum of the PBS buffer was used as control. The spectra were averaged by repeating five times scan. Fluorescence and quenching measurements. The solutions of enzymes were diluted into buffer A (50 mM potassium phosphate, pH 7.5 and 2 mM EDTA) and buffer B (50 mM potassium phosphate, pH 7.5, 2 mM EDTA and urea) to prepare samples, with a final protein concentration of 1 µmol/L each. The concentrations of urea were 2 M and 6 M. The mixture was incubated overnight at room temperature ensuring the equilibrium at any urea concentrations. The fluorescence spectra were recorded on F-7000 spectrophotometer in a 1 cm path length cuvette. Emission spectra of the tryptophan were obtained using excitation wavelengths of 295 nm and recorded for wavelength ranging from 300 to 400 nm. Excitation and emission slits were set at 5 nm with a scan speed of 1200 nm/min. For all the samples, fluorescence spectra were corrected by subscribing the background fluorescence. A baseline control was realized for each experiment with a series of samples containing various concentrations of urea. Fluorescent quenching experiments were performed by adding the solutions of acrylamide to the samples. A aliquot of acrylamide solution was added to 2 mL of the samples, the solutions were then incubated for 5 min before recording the emission spectra. Encapsulation of DAAO&CAT within a coordination polymer. 20 mg of DAAO&CAT was dissolved in 10 ml of deionized water, then 2-methylimidazole (10 20


Page 21 of 31

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


ml, 300 mM, pH 10) was added. Zinc acetate dehydrate was dissolved in deionized water to prepare a separate solution (35 mM, 25 ml).42,47 The two solutions were mixed and then agitated for 10 s. Aging for the resulting solution was carried out at 4°C for 12 h. The mixtures were then centrifuged (4 °C) for 10 min at 6000g and the supernatants were removed. Typically, six washes for the obtained precipitate were performed, with fresh PBS buffer (pH 8, 50 mM) added each time to remove unencapsulated DAAO&CAT. The concentrations of DAAO&CAT in the solutions were determined by Bradford assay,48 bovine serum albumin (BSA) was used as the standard protein. By detecting the protein in the original DAAO&CAT and washing solutions, the amount of DAAO&CAT encapsulated within the coordination polymer was determined. Average values were obtained from triplicate measurements of three encapsulation operations. The DAAO&CAT encapsulation efficiency was 91.3±2.7%. The enzyme loading was 0.39±0.015 mg DAAO&CAT/mg CP. Enzyme assays. Protein concentrations were measured by the bicinchoninic acid (BCA) assay. Bovine serum albumin (BSA) was used as the standard protein. Ten concentrations of BSA were assayed. The absorbance was plotted, and a best-fit linear line through the points was obtained and used as the calibration line. The enzyme kinetics for DAAO and DAAO&CAT was investigated at 30 °C. DAAO and DAAO&CAT were separately dissolved in 25 ml PBS buffer (50 mM, pH 8.0) to prepare enzyme solutions, and the protein concentrations were 0.002 mg/ml. D-alanine was used as the substrate, with a concentration range from 0.5 to 40 mM. The D-alanine solution was prepared by dissolving D-alanine in the PBS buffer. The concentrations of the substrate D-alanine and the product pyruvate were monitored by HPLC (Shimadzu LC-10A) with a C18 column (Diamonsil C18 250 x 4.6 mm, 5 µm). The mobile phase consisted of 0.05 M PBS, pH 2.50 and methanol (95:5 v:v) at a 21



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 22 of 31

flow rate of 0.8 mL/min and the injection volume was 20 uL. The retention times for D-alanine and pyruvate were 3.7 min and 4.8 min, respectively. Detection was performed at 220 nm. All the solutions were filtered using a 0.22 um polycarbonate membrane (Millipore) prior to the injection. The initial rate of reaction at each substrate concentration was determined by measuring the change of substrate concentration every 60 s for 5 min. At each substrate concentration, triplicate measurements were performed and averaged data were obtained. Thus for a certain substrate concentration, 15 data points were measured to determine the initial rate of reaction. For each enzyme, eight substrate concentrations were assayed, and total 120 data points were obtained for the plots of the initial rate of reaction (V0) versus substrate concentration [S]. Kinetics parameters were determined by fitting the Michaelis–Menten equation to the initial rates of reactions. For the activity assay of the encapsulated DAAO&CAT, the CP/DAAO&CAT composite was dispersed in PBS buffer (50 mM, pH 8.0) with an enzyme concentration 0.5 mg/ml. The specific activity of the enzymes was calculated by measuring the formation of pyruvate concentration in the medium, following the methods described by Biagi et al.49 One unit of enzyme activity was expressed U mg-1 enzyme. For terminating the reactions catalyzed by the free enzymes, the sample tubes were immediately transferred to the water bath operating at 100 °C. For terminating the reactions catalyzed by the encapsulated DAAO&CAT, the sample tubes were immediately transferred to liquid nitrogen for 10 min, and then thawing the samples in ice water. For qualitative comparison between DAAO&CAT and DAAO for catalyzing the 22


Page 23 of 31

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


deamination of D-alanine by showing the colour change, the reaction conditions were: the enzyme concentrations were DAAO 0.5 mg/mL and DAAO&CAT 0.5 mg/mL; D-alanine (13 mM) was dissolved in 1 ml PBS buffer (pH 8.0, 50 mM). The reactions were carried out at 30 0C for 5 min. 100 µl trichloroacetic acid solution (20 % w/v) was then added to the tubes (200 µl) to terminate the reactions. The solutions were centrifuged. 300 µl 2,4-dinitrophenylhydrazine (DNPH) solution (0.1 mM) was added to the supernatants, incubated at 37 0C for 10 min, and then 600 µl NaOH (1.5 M) was added. After 10 min at 37 0C, the color changes were observed.

■ ASSOCIATED CONTENT Supporting Information Table S1. List of oligonucleotides and genes for IntC-link and link-intN. Table S2. Molecular weight for various proteins. Scheme 1, Figures S1−S9. AUTHOR INFORMATION Corresponding Author

* E-mail: [email protected] Notes †

K. D and J. Z. equally contributed to this work.

The authors declare no competing financial interest. ACKNOWLEDGEMENTS We are grateful to Dr. Paul Xiangqing Liu (Department of Biochemistry & Molecular Biology, Dalhousie University) for providing the C-214 plasmid, Dr. Yue Feng (Beijing University of Chemical Technology) for the purification of splicing product. This work was supported by the National Science Foundation of China (Grant numbers 21376021, 21576018) and the China Postdoctoral Science Foundation (Grant number 2016M591052). 23



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 24 of 31

REFERENCES (1) Chung,

J., Hwang, E. T., Kim, J. H., Kim, B. C., and Gu, M. B. (2014). Modular

multi-enzyme cascade process using highly stabilized enzyme microbeads. Green. Chem. 16, 1163-1167; Redeker, E.S., Ta, D. T., Cortens, D., Billen, B., Guedens, W., and Adriaensens, P. (2013). Protein Engineering For Directed Immobilization. Bioconjugate Chem. 24, 1761−1777. (2) Kang,

W., Liu, J., Wang, J., Nie, Y., Guo, Z., and Xia, J. (2014). Cascade

Biocatalysis by Multienzyme–Nanoparticle Assemblies. Bioconjugate Chem. 25, 1387–1394; Qi, P., You, C., Zhang, Y.-H. P. (2014). One-Pot Enzymatic Conversion of Sucrose to Synthetic Amylose by using Enzyme Cascades. ACS Catal. 4, 1311–1317; Classen, T., Korpak, M., Schölzel, M., and Pietruszka, J. (2014). Stereoselective Enzyme Cascades: An Efficient Synthesis of Chiral γ-Butyrolactones. ACS Catal. 4, 1321–1331. (3) Kumar,

A. K., and Goswami, P. (2009). Dissociation and reconstitution studies of

a broad substrate specific multimeric alcohol oxidase protein produced by Aspergillus terreus. Biochem. 145, 259-265. (4) Prakash,

K., Prajapati, S., Ahmad, A., Jain, S. K.,and Bhakuni, V.(2002).Unique

oligomeric intermediates of bovine liver catalase. Prot. Sci. 1, 46-57 (5) Barzegar,

A., Moosavi-Movahedi, A. A., Pedersen, J. Z., and Miroliaei, M. (2009).

Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations. Enzyme Microb.Technol. 45(2), 73-79. (6) Becerra,

M., Cerdan, E., and Siso, M. I. G. (1998). Micro-scale purification of

β-galactosidase from Kluyveromyces lactis reveals that dimeric and tetrameric forms are active. Biotechnol. Tech. 12, 253-256. 24


Page 25 of 31

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


(7) Pollegioni,

L., and Molla, G. (2011). New biotech applications from evolved

D-amino acid oxidases. Trends. Biotechnol. 29, 276-283. (8) Pollegioni,

L., Caldinelli, L., Molla, G., Sacchi, S., and Pilone, M. S. (2004).

Catalytic Properties of d‐Amino Acid Oxidase in Cephalosporin C Bioconversion: A Comparison between Proteins from Different Sources. Biotechnol. Prog. 20, 467-473. (9) Upadhya,

R., and Bhat, S. G. (2000). Stabilization of D‐amino acid oxidase and

catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of α‐ketoacids. Biotechnol. Bioeng. 68, 430-436. (10)

Pollegioni, L.; Molla, G.; Sacchi, S.; Rosini, E.; Verga, R.; Pilone, M. S. (2008).

Properties and applications of microbial D-amino acid oxidases: current state and perspectives. Appl. Microbiol. Biotechnol. 78, 1–16. (11)

Chen, Y.J., Goldberg, S.L., Hanson, R.L., Parker, W.L., Gill, I., Tully, T.P.,

Montana, M.A., Goswami, A., and Patel, R.N. (2011). Enzymatic preparation of an (S)-amino acid from a racemic amino acid. Org. Process Res. Dev. 15, 241–248. (12)

Truppo, M. D., Turner, N. J., and Rozzell, J. D. (2009). Efficient kinetic

resolution of racemic amines using a transaminase in combination with an amino acid oxidase. Chem. Commun. 16, 2127-2129. (13)

López-Gallego, F., Betancor, L., Mateo, C., Hidalgo, A., Alonso-Morales, N.,

Dellamora-Ortiz, G ,Guisán, J. M.,and Fernández-Lafuente, R. (2005). Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports. J. Biotechnol. 119, 70-75. (14)

Parkin, K., and Hultin, H. O. (1979). Immobilization and characterization of D–

amino acid oxidase. Biotechnol. Bioeng. 21, 939-953. (15)

Zhao, Y., Zhu, Y., and Fu, J. (2014). Manageable cytotoxicity of nanocapsules

immobilizing D-amino acid oxidase via exogenous administration of nontoxic 25



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 26 of 31

prodrug. Appl. Surf. Sci.293, 109-115. (16)

Yoshimoto, M., Yamasaki, M., Okamoto, M., Umakoshi, H., and Kuboi, R.

(2013). Oligolamellar vesicles for covalent immobilization and stabilization of d-amino acid oxidase. Enzym.Microb. Technol. 52, 13-19. (17)

Mujawar, S. K., Kotha, A., Rajan, C. R., Ponrathnam, S., and Shewale, J. G.

(1999).

Development of

tailor-made

glycidyl

methacrylate-divinyl

benzene

copolymer for immobilization of D-amino acid oxidase from Aspergillus species strain 020 and its application in the bioconversion of cephalosporin C. J. Biotechnol. 75, 11-22. (18)

Mu, X., Qiao, J., Qi, L., Liu, Y., and Ma, H. (2014). Construction of a D-amino

acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors. Appl. Mater. Inter. 6, 12979-12987. (19)

Bolivar, J. M., and Nidetzky, B. (2012). Oriented and selective enzyme

immobilization on functionalized silica carrier using the cationic binding module Zbasic2: design of a heterogeneous D ‐ amino acid oxidase catalyst on porous glass. Biotechnol. Bioeng. 109, 1490-1498. (20)

Wong, K. S., Fong, W. P., and Tsang, P. W. K. (2011). Entrapment of a

Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C. Eng. Life. Sci. 11, 491-495. (21)

Wilson, L., Betancor, L., Fernández-Lorente, G., Fuentes, M., Hidalgo, A.,

Guisán, J. M., Pessela, B.C. C., and Fernández-Lafuente, R. (2004). Cross-linked aggregates of multimeric enzymes: a simple and efficient methodology to stabilize their quaternary structure. Biomacromolecules 5, 814-817. (22)

Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., 26


Page 27 of 31

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


Evans, C., and Srere, P. A. (1994). Preparation and kinetic characterization of a fusion protein

of

yeast

mitochondrial

citrate

synthase

and

malate

dehydrogenase. Biochemistry 33, 11692-11698. (23)

Fernandez-Lafuente, R. (2009). Stabilization of multimeric enzymes: strategies

to prevent subunit dissociation. Enzyme Microb. Technol. 45, 405-418. (24)

Xu, M. Q., Southworth, M. W., Mersha, F. B., Hornstra, L. J., and Perler, F. B.

(1993). In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell 75 1371-1377. (25)

Vila-Perelló, M., and Muir, T. W. (2010). Biological applications of protein

splicing. Cell 143, 191-200. (26)

Aranko, A. S., Oeemig, J. S., Kajander, T., and Iwaï, H. (2013). Intermolecular

domain swapping induces intein-mediated protein alternative splicing. Nat. Chem. Biol. 9, 616-622. (27)

Liu, Z., Frutos, S., Bick, M. J., Vila-Perelló, M., Debelouchina, G. T., Darst, S.

A., and Muir, T. W. (2014). Structure of the branched intermediate in protein splicing. Proc. Natl. Acad. Sci. U. S. A. 111, 8422-8427. (28)

Böcker, J. K., Friedel, K., Matern, J. C., Bachmann, A. L., and Mootz, H. D.

(2015). Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides. Angew. Chem. Int. Edit. 54, 2116-2120. (29)

Deschuyteneer, G., Garcia, S., Michiels, B., Baudoux, B., Degand, H.,

Morsomme, P., and Soumillion, P. (2010). Intein-mediated cyclization of randomized peptides in the periplasm of Escherichia coli and their extracellular secretion. ACS. Chem. Biol. 5, 691-700. (30)

Volkmann, G., Murphy, P. W., Rowland, E. E., Cronan, J. E., Liu, X. Q., Blouin,

C., and Byers, D. M. (2010). Intein-mediated cyclization of bacterial acyl carrier 27



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 28 of 31

protein stabilizes its folded conformation but does not abolish function. J. Biol. Chem. 285, 8605-8614. (31)

Yamazaki, T., Otomo, T., Oda, N., Kyogoku, Y., Uegaki, K., Ito, N., Ishino, Y.,

and Nakamura, H. (1998). Segmental isotope labeling for protein NMR using peptide splicing. J. Am. Chem. Soc. 120, 5591-5592. (32)

Ramirez, M., Guan, D., Ugaz, V., and Chen, Z. (2013). Intein-triggered

artificial protein hydrogels that support the immobilization of bioactive proteins. J. Am. Chem. Soc. 135, 5290-5293. (33)

Xu, M. Q., Southworth, M. W., Mersha, F. B., Hornstra, L. J., and Perler, F. B.

(1993). In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell 75, 1371-1377. (34)

Shah, N. H., Vila‐Perelló, M., and Muir, T. W. (2011). Kinetic Control of

One‐Pot Trans‐Splicing Reactions by Using a Wild‐Type and Designed Split Intein. Angew. Chem., Int. Ed. 50, 6511-6515. (35)

Southworth, M. W., Adam, E., Panne, D., Byer, R., Kautz, R., and Perler, F. B.

(1998). Control of protein splicing by intein fragment reassembly. EMBO. J. 17, 918-926. (36)

Wiedemann, N., Kozjak, V., Chacinska, A., Schönfisch, B., Rospert, S., Ryan,

M. T., Pfanner, N.,and Meisinger, C. (2003). Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424, 565-571. (37)

Georlette, D., Blaise, V., Bouillenne, F., Damien, B., Thorbjarnardóttir, S. H.,

Depiereux,

E.,

Gerday,

Adenylation-dependent

C.,

Uversky,

conformation

and

V.N.,

and

unfolding

Feller,

G.

pathways

(2004). of

the

NAD+-dependent DNA ligase from the thermophile Thermus scotoductus. Biophys. J. 86, 1089-1104. 28


Page 29 of 31

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


(38)

Josephy, P. D., Eling, T., and Mason, R. P. (1982). The horseradish

peroxidase-catalyzed oxidation of 3, 5, 3', 5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates. J. Biol. Chem.257, 3669-3675. (39)

Liu, Z., Frutos, S., Bick, M. J., Vila-Perelló, M., Debelouchina, G. T., Darst, S.

A., and Muir, T. W. (2014). Structure of the branched intermediate in protein splicing. Proc. Natl. Acad. Sci. 111, 8422-8427. (40)

Böcker, J. K., Friedel, K., Matern, J. C., Bachmann, A. L., and Mootz, H. D.

(2015). Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides. Angew. Chem. Int. Edit. 54, 2116-2120. (41)

Rocha-Martin, J., Velasco-Lozano, S., Guisán, J. M., and López-Gallego, F.

(2014). Oxidation of phenolic compounds catalyzed by immobilized multi-enzyme systems with integrated hydrogen peroxide production. Green. Chem.16, 303-311. (42)

Liang, K.; Ricco,

R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.;

Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C.J.; et al. (2015). Biomimetic mineralization

of

metal-organic

frameworks

as

protective

coatings

for

biomacromolecules. Nat. Commun. 6, 7240 (43)

Li, S. B.; Dharmarwardana, M.; Welch, R. P.; Ren, Y. X.; Thompson, C. M.;

Smaldone, R. A.; Gassensmith, J. J. (2016). Template-Directed Synthesis of Porous and Protective Core–Shell Bionanoparticles. Angew. Chem., Int. Ed. 55, 10691– 10696. (44)

Liang, K.; Coghlan, C. J.; Bell, S.G.; Kirby, Doonan, C.J.; Falcaro, P. (2016).

Enzyme encapsulation in zeolitic imidazolate frameworks: a comparison between controlled co-precipitation and biomimetic minerali. Chem. Commun. 52, 473–476. (45)

Molina, D.R., Comamala, D.M., Gabilondo， I. I. (2011). Metallo-organic

system for the encapsulation and release of compounds of interest, method for 29



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 30 of 31

obtaining same and uses thereof. Patent US 0064775. (46)

Nowakowski, A. B., Wobig, W. J., and Petering, D. H. (2014). Native

SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics 6, 1068-1078. (47)

Lyu, F., Zhang, Y., Zare, R. N., Ge, J., and Liu, Z. (2014). One-pot synthesis of

protein-embedded metal–organic frameworks with enhanced biological activities. Nano Lett.14, 5761-5765. (48)

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (49)

Biagi, S., Ghimenti, S., Onor, M., and Bramanti, E. (2012). Simultaneous

determination of lactate and pyruvate in human sweat using reversed‐phase high‐ performance

liquid

chromatography:

a

noninvasive

approach.

Biomed.

Chromatogr. 26, 1408-1415.

30


Page 31 of 31

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


244x156mm (120 x 120 DPI)


Specific Ligation of Two Multimeric Enzymes with Native Peptides and

Recommend Documents