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Formation of nitrile hydratase CLEAs in mesoporous onion-like silica: preparation and catalytic properties Jing Gao, Qi Wang, Yanjun Jiang, Junkai Gao, Zhihua Liu, liya Zhou, and Yufei Zhang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/ie503018m • Publication Date (Web): 15 Dec 2014 Downloaded from http://pubs.acs.org on December 21, 2014
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Industrial & Engineering Chemistry Research
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Formation of nitrile hydratase CLEAs in mesoporous onion-like silica: preparation and catalytic properties
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Jing Gaoa, Qi Wanga, Yanjun Jianga*, Junkai Gaob, Zhihua Liua, Liya Zhoua, Yufei
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Zhangc
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a. School of Chemical Engineering and Technology, Hebei University of Technology,
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7 8 9 10 11
Tianjin, 300130, China. b. School of Energy and Environmental Engineering, Hebei University of Technology, Tianjin, 300130, China. c. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
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* Corresponding author: Fax: +86-22-60204294; Tel: +86-22-60204945;
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E-mail address:
[email protected] (Yanjun Jiang)
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Abstract:
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Nitrile
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(NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a
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cross-linker through the carrier-bound CLEAs method. The effect of preparation
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parameters on the recovery of enzyme activity was investigated. The properties such
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as pH, thermal, and storage stability and kinetic parameters of NHase-CLEAs@MOS
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were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g.
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Under
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NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were
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improved significantly compared to the NHase@MOS prepared by physical
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adsorption and free NHase. This work demonstrated that the mesoporous onion-like
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silica can be efficiently employed as host materials for NHase immobilization and the
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carrier-bound CLEAs method can lead to enhanced activity and stability of the
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immobilized enzymes.
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Keywords:
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immobilization; mesoporous silica; dextran polyaldehyde
hydratase
CLEAs
optimized
was
conditions,
cross-linked
enzyme
formed
the
in
mesoporous
maximum
aggregates;
activity
nitrile
onion-like
silica
recovery
hydratase;
of
enzyme
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1. Introduction
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Nitrile hydratase (NHase; EC 4.2.1.84) is a class of mononuclear iron or cobalt
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enzymes that catalyze the hydration of nitriles to their corresponding amides.1 NHases
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are widely used in the industrial production of acrylamide and nicotinamide, and they
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can also be used in environmental remediation for the conversion of nitrile wastes to
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less toxic amides.2-6 However, the industrial use of NHase is often limited by the low
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operational stability, highly sensitive to the change of environment and poor recovery
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of the free NHase. To overcome these drawbacks, immobilization of NHase on a
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suitable support is a promising strategy, which not only improves the operational
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stability of the enzyme, but also facilitates the efficient recovery and reuse.7,8 Until
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now, different methods including adsorption or covalent binding to a carrier,
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encapsulation and entrapment or cross-linking have been developed to immobilize
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enzymes.7,8 However, each immobilization method has its own advantages and
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disadvantages and a “perfect” universally applicable method for immobilizing
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enzymes is not available. NHase ES-NHT-118, obtained from E. coli strain that
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carries the cloned nitrile hydratase gene, is an important enzyme in converting nitriles
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to amides under physiological conditions. Developing an efficient immobilized
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method for this kind of NHase is the prerequisite for its industrial applications (i.e.
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acrylamide production).
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In recent years, a new approach which combines the advantages of physical
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adsorption with cross-linked enzyme aggregates (CLEAs) was developed. The
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so-called carrier-bound CLEAs can be prepared easily by the following process:
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enzymes are adsorbed on porous supports firstly, aggregated and then cross-linked,
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resulting in the formation of CLEAs in the pores.9-11 For example, Kim and
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co-workers prepared α-chymotrypsin and lipase CLEAs in hierarchically-ordered
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mesocellular mesoporous silica (HMMS) through “ship-in-a-bottle” approach, and the
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enzymes showed high loading capacity and increased stability.12 As an extension of
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this approach, magnetically-separable and highly-stable carrier-bound CLEAs
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systems were also prepared. Besides the advantages of high enzyme loadings and
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stability, facile magnetic separation of these immobilized enzyme systems facilitated
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their repeated usages.13 Hartmann's group reported the successful preparation of
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cross-linked chloroperoxidase and glucose oxidase aggregates in mesocellular foams.
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Compared to the immobilized enzymes on the same support through physical
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adsorption, the carrier-bound CLEAs systems showed increased operational
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stability.14,15 This approach was also used to prepare an immobilized bi-enzymatic
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system and the resulting bi-enzymatic system can be successfully used in a
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continuously-operating fixed bed reactor and the leaching of the enzymes can be
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suppressed.16 Therefore, the preparation of CLEAs in the pores of a suitable support is
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a most promising method for enzyme immobilization.9, 17
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Recently, a novel mesoporous onion-like silica (MOS) was synthesized and used as
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support for the preparation of nanoscale enzyme reactors by cross-linking adsorbed 4
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lipase. The highly ordered onion-like multilayer and highly curved pore structure of
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the MOS was effective in improving the lipase stability.17 Further studies of enzyme
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immobilization in this novel porous materials via carrier-bound CLEAs method and
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systematic investigation of the resulting biocatalyst are currently still required.
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Thus, in this study, MOS was synthesized according to the previous report17 and the
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NHase ES-NHT-118 was immobilized in MOS by adsorption (named NHase@MOS).
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To avoid the leaching problem and improve the properties of NHase, dextran
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polyaldehyde (DP) was used to cross-link the adsorbed NHase and then cross-linked
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enzyme aggregates in the MOS was obtained (named NHase-CLEAs@MOS). This is
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the first time that successful immobilization of NHase by using the carrier-bound
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CLEAs method. The preparation conditions were optimized and the effects of pH and
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temperature on the activity of NHase-CLEAs@MOS were investigated. The pH,
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thermal and storage stabilities of NHase-CLEAs@MOS were also investigated.
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2. Experimental
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2.1 Materials
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Nitrile hydratase ES-NHT-118 (NHase, EC 4.2.1.84) from E. coli strain that carried
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the cloned nitrile hydratase gene was purchased from Hangzhou Biosci Biotech Co.
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(China). Poly(ethlyene glycol)-block-poly-(propylene glycol)-block-poly(ethylene
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glycol) triblock copolymer (EO)20(PO)70(EO)20 (denoted as P123, Mn=5800),
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tetraethyl orthosilicate (TEOS) and 1,3,5-Trimethylbenzene (TMB, 98%) were
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purchased from Sigma-Aldrich (America). SBA-15 was purchased from Nanjing 5
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XFNANO, inc. (China). Dextran (100 kDa) and sodium metaperiodate were
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purchased from Dingguo Biotechnology Co. (China). Acrylonitrile was purchased
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from Fuchen Chemical Reagent Factory (China). All reagents were used as received
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without further purification.
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2.2 Synthesis and characterization of mesoporous onion-like silica
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Mesoporous onion-like silica (MOS) was prepared according to the reported
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procedure.17 Briefly, an amount of P123 (4 g) was dissolved in 150 mL of HCl
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solution (1.6 M) under stirring condition at room temperature. Then, 2 g of TMB was
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added and stirred for 5 h. The mixture was heated to 40 oC and then of an amount of
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TEOS (8.5 g) was added under stirring. After that, the mixture was aged at 40 oC for
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20 h under stirring condition and further aged at 100 oC under static condition for 24 h.
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The solid was filtered, washed, and calcined at 550 oC for 4 h, and then the MOS was
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obtained.
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Transmission electron microscopic (TEM) images of the MOS were obtained on a
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Tecnai G2 F20 Transmission Electron Microscope. Scanning electron microscopic
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(SEM)
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adsorption/desorption isotherms at 77 K were obtained using a Micromeritic
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ASAP2020M+C Physisorption Analyzer. Pore size distribution was calculated using
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the BJH method. Fourier-transform infrared (FT-IR) spectra were recorded on a
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Bruker Vector 22 FT-IR spectrophotometer using KBr pellets method. The
images
were
obtained
on
a
FEI
NanoSEM450
microscope.
N2
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thermogravimetric
analysis
(TGA)
was
performed
on
a
SDT
Q600
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Thermogravimetric Analyzer under air atmosphere and heating rate of 10 oC/min.
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2.3 Preparation of immobilized NHase
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MOS (50 mg) was mixed with 10 mL of NHase solution (50 mM sodium phosphate
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buffer, PBS, pH 7.0) in a centrifuge tube and then incubated in a water bath (25 oC)
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for several hours under shaking condition (200 rpm). After that, the solid was
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separated from the supernatant liquid by centrifugation. The obtained solid (referred
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to as NHase@MOS) was washed with PBS (50 mM, pH 7.0) for three times. The
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concentration of NHase in the solutions was measured by Bradford assay. Typically,
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1mL NHase solution was mixed with 5 mL Bradford reagent and allowed to stand for
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5 min before measuring its absorbance at 595 nm. The final enzyme loading on the
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MOS was calculated from the difference between the initial NHase amount and the
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residual amount of NHase in the washing solutions and supernatant. For preparation
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of NHase-CLEAs@MOS, NHase@MOS was incubated in DP solution for a certain
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time at a low temperature (
