Preparation and characterization of a highly stable phenoxazinone synthase nanogel
© The Author(s) 2016
Received: 22 September 2015
Accepted: 10 May 2016
Published: 28 May 2016
Phenoxazinone synthase (PHS) is a laccase-like multicopper oxidase originating from Streptomyces with great industrial application potential. In this paper, we prepared the PHS nanogel retaining 82 % of its initial activity by aqueous in situ polymerization at pH 9.3.
The average diameter of the PHS nanogel was 50.8 nm based on dynamic light scattering (DLS) analysis. Fluorescence analysis indicated the impressive preservation of the enzyme molecular structure upon modification. The PHS nanogel exhibited the most activity at pH 4.0–4.5 and 50 °C while the corresponding values were pH 4.5 and 40 °C for the native PHS. The K m and V max of the PHS nanogel were found to be 0.052 mM and 0.018 mM/min, whereas those of the native PHS were 0.077 mM and 0.021 mM/min, respectively. In addition, the PHS nanogel possessed higher thermal and storage stability and solvent tolerance compared with the native one. The half-life of the PHS nanogel was 1.71 h and multiplied around ninefold compared to 0.19 h for the native one.
In summary, the PHS nanogel could be a promising biocatalyst in industry.
KeywordsPhenoxazinone synthase Laccase Nanogel Stability Solvent resistance
Phenoxazinone synthase (PHS, EC 22.214.171.124) is a bacterial laccase-like multicopper oxidase firstly described by Katz and Weissbach . As a key enzyme for actinomycin D biosynthesis in Streptomyces, the properties of PHS were preliminarily characterized originally by Golub and Nishimura . They found it can catalyze oxidation of catechols, ferrocyanide, and ethylenic thiols, in addition to o-aminophenols, which was similar to laccase. In general, PHS exists in a hexameric form which exhibits the most activity . In consideration of its catalytic properties, PHS is a promising enzyme for use in antibiotics production, dye synthesis, bio-bleaching, and bio-detoxication [4–7].
Owing to lower stability, enzymes usually fail to meet the need of industrial processes. For a long time, chemical modification of key groups has enabled enzyme improvement in terms of stability and other features [8–10]. Unlike the other methods, chemical modification can unlimitedly alter side chain of amino acid structures without the need of sequence or structure information . Chemical modification might strengthen the intrinsic rigidity of the molecule to enhance pH and temperature stability and organic solvent tolerance [8, 12].
In recent years, enzyme modification on a nanoscale is drawing more and more attention for its ability to confer higher activity and stability [13, 14]. The soluble single-enzyme nanoparticles (SENs) of α-chymotrypsin and trypsin have been prepared by surrounding enzyme molecule with a nanometer thick porous composite organic/inorganic network, and exhibited impressive stability with minimal substrate mass-transfer limitation . After that, the SENs has been embedded into nanoporous silica and showed higher operational stability . Besides, several similar enzyme nanogels involving horseradish peroxidase, lipase, carbonic anhydrase and laccase have been synthesized by using an innovative aqueous in situ polymerization with excellent thermal stability and tolerance resistance [17–21]. The possible mechanism for improving stability has also been proposed by molecular simulation [22, 23].
In the present study, for the purpose of improving the properties, we prepared the PHS nanogel via in situ polymerization. The resultant PHS nanogel was analyzed by SEC, and fluorescence analysis. Subsequently, kinetic parameters, thermal and storage stability, and solvent tolerance were also characterized in detail.
Results and discussion
Effect of pH on the modification
Effect of concentration of acrylamide on PHS nanogel preparation
DLS and fluorescence analysis
Optimum pH and temperature
The temperature profiles of the native PHS and PHS nanogel were also examined over a temperature range from 25 to 75 °C. As can be seen from Fig. 5b, the native PHS reached its maximum activity at 40 °C, whereas it shifted to 50 °C for the nanogel. The shift in optimum temperature was attributed to the change on conformational flexibility as a result of formation of covalent bonds between the enzyme and the polyacrylamide gel .
Kinetic parameters of the native PHS and PHS nanogel
v −1 = 3.59[S]−1 + 46.73 (R 2 = 0.9981)
v −1 = 2.87[S]−1 + 55.11 (R 2 = 0.9984)
PHS was prepared according to the previous publication . N-Acryloxysuccinimide (NAS), 2, 2′-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2, 4, 6-trinitrobenzenesulfonic acid solution (TNBS) were purchased from Sigma-Aldrich (Shanghai, China). Tetramethylethylenediamine (TEMED), acrylamide, ammonium persulfate and trehalose were supplied by Sinophar Chemical Reagent Co., Ltd (Shanghai, China). All other chemicals used were of analytical grade.
The preparation of PHS nanogel
Determination of modified amino group
The sulfonate group of TNBS can react specifically with the free amino groups of proteins and the resulting derivatives can be determined spectrophotometrically. TNBS method is usually used for the determination of free amino groups in proteins [46, 47]. In this paper, the modified amino group in the PHS preparation was determined by using the TNBS method, and the modification yield was defined as the ratio of modified amino groups in protein.
The DLS analysis of the native PHS and PHS nanogel was conducted at 25 °C on a Brookhaven BI-200SM laser light scattering system with a 90° scattering angle.
The fluorescence analyses of the native PHS and PHS nanogel excited at 285 nm were recorded from 300 to 550 nm with a Shimadzu RF-5301 PC spectrofluorometer.
Determination of PHS activity
The native PHS and PHS nanogel activity was determined spectrophotometrically by monitoring the increase in absorbance at 420 nm of a reaction mixture containing 0.5 mM ABTS in 0.1 M sodium acetate buffer (pH 4.5) and a suitable amount of enzyme at 25 °C . One unit of PHS activity was defined as the amount of enzyme oxidizing 1 μmol of ABTS per minute (ε420 = 36 mM−1 cm−1).
Optimum pH and temperature
To investigate the optimum pH and temperature of the native PHS and PHS nanogel, the activity of the native PHS and PHS nanogel was measured using ABTS as substrate at pH (3.0–8.0) and temperature (25–75 °C), respectively.
The kinetic parameters, K m and V max, of the native PHS and PHS nanogel were calculated by the Lineweaver–Burk plot. Reactions were conducted based on the determination of activity method using 0.05–0.5 mM ABTS.
The native PHS and PHS nanogel stabilizing against thermal denaturation were tested in acetate buffer (100 mM, pH 4.5) at 60 °C and the activity was determined after sampling periodically as described above. The residual activity was expressed as the percentage with respect to initial activity.
The investigations into solvent tolerance of the native PHS and PHS nanogel were carried out by incubating in different organic solvents at 30 °C for 1 h. Then the activities were assayed as described above.
In this paper, a designed nanogel prepared by aqueous in situ polymerization at pH 9.3, which could retain 82 % of PHS activity was introduced. The average diameter of the PHS nanogel was 50.8 nm based on dynamic light scattering analysis. Fluorescence analysis indicated the impressive preservation of the enzyme molecular structure upon modification. The PHS nanogel exhibited the most activity at pH 4.0–4.5 and 50 °C while the corresponding values were pH 4.5 and 40 °C for the native PHS. The K m and V max of the PHS nanogel were found to be 0.052 mM and 0.018 mM/min, whereas those of the native PHS were 0.077 mM and 0.021 mM/min, respectively. In addition, the PHS nanogel had possessed higher thermal and storage stability and solvent tolerance compared with the native one. The half-life of the PHS nanogel was 1.71 h and had multiplied around ninefold compared to 0.19 h for the native one.
It is the first investigation into the nanogel preparation and characterization of PHS (phenoxazinone synthase) originated from Streptomyces in this paper. Based on the enzymatic properties were characterized in detail, results showed that the resultant PHS nanogel have indicated higher thermal and storage stability and solvent resistance. As a result, the PHS nanogel could be a promising biocatalyst in industry.
2, 2′-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt
dynamic light scattering
2, 4, 6-trinitrobenzenesulfonic acid solution
HHJ carried the literature study, designing part, designing of schemes as well as drafting of the manuscript. ZG carried the preparation of nanogel. YYM and CZ contributed characterization of nanogel. HHJ, CMW, HZ and PW conceived the project. All authors read and approved the final manuscript.
The research was supported financially by NSFC (20906048), the State Key Basic Research and Development Plan of China (2013CB733500), National Key Technology R&D Program (2014BAC33B00), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), PCSIRT (IRT_14R28) and PAPD.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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