- Email: [email protected]
Creation of thermostable l -tryptophan dehydrogenase by protein engineering and its application for l -tryptophan quantification
Accepted Manuscript Creation of thermostable L-tryptophan dehydrogenase by protein engineering and its application for L-tryptophan quantification Daisuke Matsui, Yasuhisa Asano PII:
S0003-2697(18)30995-3
DOI:
https://doi.org/10.1016/j.ab.2019.05.010
Reference:
YABIO 13321
To appear in:
Analytical Biochemistry
Received Date: 28 January 2019 Revised Date:
11 May 2019
Accepted Date: 13 May 2019
Please cite this article as: D. Matsui, Y. Asano, Creation of thermostable L-tryptophan dehydrogenase by protein engineering and its application for L-tryptophan quantification, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2019.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Running title: Protein engineering for L-tryptophan quantification
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Subject Category: Enzymatic Assays and Analyses
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Creation of thermostable L-tryptophan dehydrogenase by protein engineering and its
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application for L-tryptophan quantification
Daisuke Matsui1, 2 and Yasuhisa Asano1, 2†
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University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
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939-0398, Japan
Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama
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Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural
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†
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E-mail: [email protected]
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To whom correspondence should be addressed. Tel: +81-766-56-7500; Fax: +81-766-56-2498;
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List of abbreviations
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HPLC
High performance liquid chromatography
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IPA
Indole-3-pyruvic acid
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LB
Luria-Bertani
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LeuDH
L-Leucine
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NAD+
Nicotinamide adenine dinucleotide
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NADH
Nicotinamide adenine dinucleotide – hydrogen
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NMR
Nuclear Magnetic Resonance
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NpR1275
L-Tryptophan
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PCR
Polymerase chain reaction
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PheDH
L-Phenylalanine dehydrogenase
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StaO and VioA
L-Tryptophan
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taPp
Threonine aldolase gene
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TMO
L-Tryptophan
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tnaA
Tryptophanase gene
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Tris
Tris(hydroxymethyl)aminomethane
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trpABCDE
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trpB
Tryptophan synthase beta chain gene
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TrpDH
L-Tryptophan
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dehydrogenase gene
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oxidase
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2-monooxygenase
Component operon of tryptophan biosynthetic pathway
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dehydrogenase
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dehydrogenase
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Abstract L-Tryptophan
dehydrogenase is a new NAD+-dependent amino acid dehydrogenase
discovered in Nostoc punctiforme. The enzyme is involved in scytonemin biosynthesis and is
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highly selective toward L-tryptophan. By a growth-dependent molecular evolution technique, a
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thermostable mutant enzyme was selected successfully. L-Tryptophan concentration in human
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plasma was successfully determined using the thermostable mutant of L-tryptophan
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dehydrogenase.
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1. Introduction L-Tryptophan is
an essential amino acid originally isolated from enzymatic hydrolysis of
casein and the structure was identified by Hopkins and Cole in 1902 [1]. L-Tryptophan is also
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known as an important amino acid in two biosynthetic pathways; first is the biosynthesis of
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nicotinamide adenine dinucleotide via formylkynurenine and kynurenine, and the second is the
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biosynthesis of melatonin via 5-hydroxytryptophan and serotonin. Recently, a significant
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correlation between plasma tryptophan concentration and depressive illness was reported [2]. It
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has been shown that L-tryptophan is linked to fibromyalgia [3], and L-tryptophan has recently
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been identified as a useful biomarker for diagnosing inflammatory bowel disease [4]. Because
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of these clinical implications, a simple assay method for quantification of L-tryptophan is
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expected to greatly benefit the medical community.
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L-Tryptophan
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dehydrogenase (TrpDH, EC1.4.1.19) is useful for the determination of
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L-tryptophan
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throughput clinical use. Therefore, the Asano group attempted to improve the thermal stability
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of TrpDH by utilizing new methods to develop a simple and rapid enzymatic method to
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determine L-tryptophan concentration [5]. Directed evolution is a powerful tool used to identify
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variants with desired properties such as high stability, but the process is problematic in that it
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requires screening large libraries. In order to improve the hit-rate of beneficial variants, the
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authors utilized directed evolution to obtain a mutant enzyme by the combination of random
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mutagenesis of the gene using error-prone PCR and the transformation into an L-tryptophan
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auxotroph, “growth-dependent molecular selection”.
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concentration but the enzyme is not stabile and therefore not suitable for high
In this review, we summarize how to reveal the characteristics of a new NAD+-dependent
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TrpDH of the scytonemin biosynthesis pathway in cyanobacteria Nostoc punctiforme ATCC
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29133, and how to utilize the protein engineering techniques to improve the properties of the 4
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TrpDH. Characteristics of TrpDH variant and determination of L-tryptophan in human plasma
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using the variant are also described.
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2. Materials and method
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2.1. Investigation of TrpDH from N. punctiform ATCC 29133
NpR1275 were amplified from N. punctiforme ATCC 29133 genomic DNA, and the
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tryptophan dehydrogenase activity of the recombinant enzyme was confirmed using a
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spectrophotometic assay measuring the formation of NADH [6]. To investigate substrate
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specificity, 1.0 mM each of twenty proteolytic amino acids and D-tryptophan were used as
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substrates [5].
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2.2. Enzyme selections by the growth-dependent molecular evolution technique
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2.2.1. Selection of broad specificity amino acid racemase for modification of substrate
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specificity [7]
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Random mutagenesis on a gene of broad specificity, namely amino acid racemase from
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Pseudomonas putida IFO 12996, was carried out by error-prone PCR method, and Escherichia
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coli JM101: ∆tnaA, ∆trpABCDE harboring the mutated gene were grown in M9 medium
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containing 5 mg/L D-tryptophan and 50 mg/L casamino acids at 37°C for 16 h. The activities of
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colony-forming mutants were compared by means of a colorimetric assay mixture which
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consists 10 mM Tris-HCl (pH 8.5), 1 U/mL D-amino acid oxidase, 100 U/mL peroxidase, 5 mM
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phenol and 1 mM 4-amino antipirine.
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2.2.2. Selection of L-threonine aldolase for increase of activity [8]
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Cells containing a threonine aldolase gene library were grown in LB medium containing 150 5
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µg/mL ampicillin, and serial dilutions were plated in parallel onto minimal M9G plates lacking
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glycine but containing 10 ng/mL or 50 ng/mL tetracycline and 0.2 mM L-threonine or 0.2 mM
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L-allothreonine. The conversion
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monitored by UV spectroscopy (∆ε279 nm=1400 M-1cm-1).
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2.2.3. Identification of L-threonine aldolase [9]
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of L-β-threo-phenylserine to benzaldehyde and glycine was
P. putida KT2440 ∆taPp-carrying plasmids with threonine aldolase genes were cultivated overnight at 30 °C in 5 mL LB medium containing 50 µg/mL kanamycin, and a final cell
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concentration corresponding to an optical density of 0.05 at 600 nm was used to inoculate 5-15
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mL of minimal medium M9 with different concentrations of DL-threo-β-phenylserine as sole
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carbon source containing the corresponding antibiotic. The threonine aldolase activity towards
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threonine was measured spectrophotometrically using UV absorbance at 340 nm by coupling
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the reduction of the formed acetaldehyde with yeast alcohol dehydrogenase at 25°C.
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2.2.4. Selection of L-tryptophan dehydrogenase for increase of stability and activity [5]
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E. coli ∆trpB was transformed with the pUC19 carrying random mutated TrpDH gene, and E. coli ∆trpB harboring the mutated gene were grown in 5.0 mL of a modified M9 medium
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containing 5.0 mg/L indole-3-pyruvic acid, 100 µg/mL ampicillin, and 0.5 mM
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isopropyl-β-D-thiogalactopyranoside at 30oC for 16 h. TrpDH activity of the crude extract was
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determined by monitoring the reductionof β-NAD+ at 340 nm at 30◦C.
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2.3 Instrumental and enzymatic assays for L-tryptophan quantification
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2.3.1. High-performance liquid chromatography (HPLC) [10]
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HPLC analysis was performed using a Waters 600E pump, gradient controller (Waters
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Chromatography Corp., Milford, MA, USA), and 100 mm x 4.7 mm I.D., packed with
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Partisphere 5 µm C18 (Whatman, Clifton, NJ, USA). Separations were achieved at ambient 6
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temperature by isocratic elution at a flow-rate of 1.0 mL/min, and the quantification were
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carried out with a Waters 470 scanning fluorescence detector connected on line with a Waters
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994 programmable photodiode array detector.
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2.3.2. Electrospray ionization tandem mass spectrometry [11]
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Electrospray ionization tandem mass spectrometry analysis was carried out using an LC
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system (LC Packings Ultimate capillary HPLC system, Amsterdam, The Netherlands), a 150 x
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0.5 mm column packed with 3 µm C18 particles (ODS AQ, YMC), and a PE-Sciex API 365
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triple quadrupole mass spectrometer (PE-Sciex, Concord, Canada) equipped with pneumatically
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assisted electrospray ionization interface (Ionspray).
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2.3.3. Flow-injection amperometric biosensor based on immobilized L-tryptophan
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2-monooxygenase [12]
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Tryptophan-2-monooxygenase from P. savastanoi was produced in E. coli using a pUC19
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expression system, and tryptophan determination was carried out in continuously aerated 100
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mM Tris buffer, pH7.8, at a flow rate 3.5 mL/min, at 25°C using the amperometric batch unit
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with a Clark electrode and flow-injection amperometric system 'MultiFerm' (YerPhI, Armenia).
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2.3.4. Colorimetric assay, coupling L-tryptophan oxidases and peroxidase [13]
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Reaction mixtures contained 20 mM Tris-HCl (pH 9.0), 0-100 µM L-tryptophan, 1 mM
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4-aminoantipyrine,1 mM phenol, 15 U/mL horseradish peroxidase (Wako), and 10 mU/mL
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tryptophan oxidase solution in a final volume of 200 µL. Absorbance was measured at 505 nm
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by the microplate reader at the end of the reaction, using an extinction coefficient of 6.4 mM-1
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cm-1 for the quinoneimine dye product.
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2.3.5. Monitoring of absorbance change at 340 nm by L-tryptophan dehydrogenase [5, 14]
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The reaction was performed in a total volume of 200 µL containing100 mM glycine-KCl-KOH buffer (pH 11.5), 2.5 mM β-NAD+, and an appropriate amount of the sample. 7
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The reaction was started by the addition of enzyme, and was followed by the absorbance at 340
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nm using a microplate spectrophotometer at 30◦C for 10-30 min. The absorbance changes for
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L-tryptophan
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enzyme from the absorbance values with enzyme in each L-tryptophan concentration. In the
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assay of L-tryptophan in human plasma, human plasma was purchased from a commercial
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source (Cosmo Bio; Tokyo, Japan).
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concentration were determined by subtracting the absorbance values without
3. Results
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3.1. Screening of TrpDH in a new biosynthesis pathway and selection of a thermostable
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variant
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3.1.1. New screening of TrpDH from the scytonemin biosynthesis pathway Various plants such as pea, spinach, and tobacco contain active L-tryptophan
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dehydrogenases (EC 1.4.1.19), which is the primary enzyme of the indolylpyruvate pathway in
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plants [15]. Because these enzymes have not been purified and the genes not cloned,
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characteristics such as substrate specificities have not been investigated.
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The indole-alkaloid scytonemin is the most common and widespread sunscreen among
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cyanobacteria; its biosynthetic route is shown in Scheme 1. In addition, it was revealed that an
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18-gene cluster (NpR1276 to NpR1259) [16] is responsible for scytonemin biosynthesis in the
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cyanobacterium N. punctiforme ATCC 29133 [17]. The scytonemin biosynthetic genes are
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highly conserved in other cyanobacteria [16]. Balskus and Walsh assigned the accepted
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functions to each gene in the scytonemin gene cluster [18], and revealed that NpR1275
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resembled L-leucine dehydrogenase (LeuDH, EC 1.4.1.9, PDB code 1LEH) from B. sphaericus
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(sequence identity 46% with LeuDH). The overexpressed protein was shown to catalyze the
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oxidative deamination of L-tryptophan (referred to here as L-tryptophan dehydrogenase
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(TrpDH)), as revealed by a spectrophotometric assay measuring the formation of NADH [6, 18]. In order to determine the substrate specificity of the protein encoded by NpR1275 in N.
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punctiforme ATCC 29133, an expression plasmid containing the gene and expressed in E. coli
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was constructed. The activity of the recombinant enzyme against 20 different of L-amino acids
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and their derivatives was measured. The purified enzyme catalyzes oxidative deamination (1.03
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U/mg) only of L-tryptophan [14]. A homology model of TrpDH with the substrate L-tryptophan
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using the structural information of LeuDH as a template was also constructed. The structure
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folded into two domains (domain I and domain II) separated by a deep cleft, which is thought to
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be the substrate binding site. The Ohshima group also cloned the homologous gene from N.
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punctiforme NIES-2108 and revealed the characteristics of its TrpDH [19]. NMR analysis of the
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hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that
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TrpDH has a pro-S (B-type) stereospecificity similar to that of the
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glutamate/leucine/phenylalanine/valine dehydrogenase family. Furthermore, the characteristics
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and thermal stability of TrpDH for application in an L-tryptophan assay was examined. TrpDH
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from N. punctiforme ATCC 29133 exhibits specific dehydrogenase activity for L-tryptophan and
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shows a low Km value (0.201 mM). Once again, the enzyme is not stabile and therefore not
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suitable for high throughput clinical use. [14]. Most of the reports on enzyme stabilization focus
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on the effect of additives such as glycerol, sorbitol, and trehalose [20]. TrpDH also shows
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activity following the addition of 20% glycerol, but other additives increase the absorbance
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spontaneously in the L-tryptophan assay. Therefore, the Asano group attempted to stabilize the
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enzyme by directed evolution [5].
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3.1.2. Selection of thermostable TrpDH by the growth-dependent molecular evolution
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technique
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Directed enzyme evolution has been used in the past two decades as a powerful tool for generating enzymes with desired properties. Enzyme variants have evolved under extreme
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conditions, such as in high temperatures, acidic and alkaline environments, and organic solvents,
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[21, 22] resulting in their improved catalytic activity and specificity for new substrates [23, 24].
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The typical library size needed for directed enzyme evolution is many orders of magnitude
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larger than the number of protein variants that can be screened in a reasonable period. The
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bottleneck for most directed enzyme evolution endeavors is the availability of a genuinely
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high-throughput screen or the ability to select for target activity.
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Numerous investigators developed the selection strategy, and the “growth-dependent molecular selection” as described below (Table 1). These strategies have become breakthrough
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techniques. The Kino group used an L-tryptophan auxotroph to modify an amino acid, leading to
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a racemase enzyme with high tryptophan racemization activity [7]. On minimal medium
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containing D-tryptophan, only the L-tryptophan auxotroph E. coli, harboring the mutant bar
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protein with increased tryptophan racemase activity, was able to convert sufficient D-tryptophan
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to L-tryptophan required for growth. The Hilvert group developed a new genetic selection
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system for threonine aldolases, where retro-aldolase activity was directly linked to cellular
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growth by simultaneous inactivation of four essential genes involved in E. coli glycine
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biosynthesis [8]. This glycine auxotrophic strain could only grow in minimal medium
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supplemented with glycine or by coupling with a glycine-liberating enzyme. Recently, the
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Gröger group developed a highly efficient selection system for L-threonine aldolase using P.
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putida KT2440 [9]. L-Threonine aldolase genes of E. coli and Saccharomyces cerevisiae and the
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D-threonine aldolase
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strain P. putida KT2440 ∆taPp, followed by cultivation on minimal medium supplemented with
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DL-threo-β-phenylserine. The
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gene of Achromobacter xylosoxidans were introduced into the selection
results demonstrate that only the selection strains with 10
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plasmid-encoded L-threonine aldolases were able to grow on this racemic amino acid specific
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medium. The Asano group applied the “growth-dependent molecular selection” to TrpDH mutant
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screening [5, 25]. The mutant library was constructed using the error-prone PCR method and E.
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coli was transformed with a disrupted L-tryptophan synthase gene (E. coli ∆trpB). In the
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screening of TrpDH mutants, the complementation of L-tryptophan auxotroph was employed as
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an indicator of increased TrpDH activity and stability. The L-tryptophan auxotroph E. coli ∆trpB,
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harboring a mutated gene encoding a TrpDH variant with higher TrpDH activity, was able to
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convert a large amount of indole-3-pyruvic acid (IPA) to L-tryptophan (Fig. 1A). TrpDH activity
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of colonies cultured on M9 modified agar plates was measured. In these variants with higher
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TrpDH activity, four amino acid substitutions (L59F, D168G, A234D, and I296N) were
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identified. In order to compare the complementation of the L-tryptophan auxotroph, E. coli
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∆trpB was transformed with plasmids containing either wild type or a variant gene insertion.
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Growth time of transformants harboring wild type and mutant genes were 0.16 h-1 and 0.27 h-1,
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respectively (Fig. 1B), which indicated that the improved properties of the mutant enzyme are
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involved in the complementation of the L-tryptophan auxotroph.
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3.2. Characteristics of TrpDH and L-tryptophan quantification using various assay
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3.2.1. Characteristics of TrpDH variants [5]
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In the variants with higher TrpDH activities than wild type, the following amino acid
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substitutions were identified: L59F/D168G/A234D/I296N (TrpDH-4mut). Single point
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mutations at each amino acid residue were introduced into the wild type enzyme, and properties
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of the mutant enzymes were compared with those of the wild type enzymes. When the activities
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were measured after 72 hours and the residual activities were calculated, the Asp168 and Ile296 11
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substitutions increased the stability of the mutant (closed triangles and closed squares in Fig. 2),
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while the enzymatic activity of TrpDH L59F and A234D increased approximately 1.77 and
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6.32-fold (an open triangle and an open square at 0 hour in Fig. 2), respectively, compared to that
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of the wild type TrpDH. It was revealed that the specific activity and stability of TrpDH-4mut
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(closed circles in Fig. 2) were higher than those of the wild type enzyme (open circles in Fig. 2).
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3.2.2. L-Tryptophan quantification using TrpDH-4mut [5]
TrpDH-4mut was used for the determination of L-tryptophan (Fig. 3A), yielding absorbance
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values that increased linearly with an increase in L-tryptophan concentration, producing a
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standard curve that was linear (open circles in Fig. 3B). Furthermore, the 0 - 30 µmol of
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L-tryptophan
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absorbance change at 340 nm was measured at 30°C for 30 min. The resulting plot was also
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linear (closed circles in Fig. 3B). The same concentration of L-tryptophan was also assayed by
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ultra-performance liquid chromatography. L-Tryptophan determination using TrpDH was
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achieved by stabilizing the enzyme using protein engineering as described in this paper.
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3.3. Instrumental and the other enzymatic assays for determining L- tryptophan
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in human plasma, was incubated with 1.0 unit of TrpDH at pH 11.5, and the
To date, various instrumental methods using high performance liquid chromatography
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(HPLC) [10] or electrospray ionization tandem mass spectrometry [11] have been developed to
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determine L-tryptophan levels in plasma (Table 2). The sensitively of these detection systems is
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high, but these methods require specialized systems and long run times. An amperometric
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biosensor with L-tryptophan 2-monooxygenase (TMO, EC 1.13.12.3) [12] and colorimetric
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assays using L-tryptophan oxidases (EC1.4.3.-) and peroxidase have been reported as enzymatic
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methods to determine L-tryptophan [13, 26].
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4. Discussion Microbial NAD+-dependent TrpDH, which catalyzes the oxidative deamination and reductive amination between L-tryptophan and IPA, was found in the scytonemin biosynthetic
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pathway of N. punctiforme ATCC29133 [6, 18]. This TrpDH exhibits high specificity toward
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L-tryptophan,
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the present study suggest that metabolism-oriented screening is a promising strategy for
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obtaining enzymes with thermostability, which are often difficult to obtain by random screening.
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The “growth-dependent selection system” was adapted for the screening of thermostable
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but its instability is a drawback for L-tryptophan determination [14]. The results in
variants. In order to efficiently obtain higher-stability TrpDH variants from N. punctiforme
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ATCC 29133 that would be useful in the determination of L-tryptophan, the Asano group
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utilized the following approach: directed evolution, involving random mutagenesis of the
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NpR1275 gene using error-prone PCR and its transformation into an L-tryptophan auxotroph, E.
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coli ∆trpB. In this screening method, the transformants harboring the mutated NpR1275 genes
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were cultivated in a medium containing the L-tryptophan precursor IPA as the main carbon
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source [5]. This is because TrpDH is a key enzyme used by the E. coli transformants to
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assimilate IPA. Using this selection method, one variant with high catalytic activity and stability
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was obtained, and was used for the development of the L-tryptophan assay. This method is one
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of only a few enzymatic L-tryptophan assays available.
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At the present time, developments in metabolomic approaches have enabled investigators to
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measure various metabolites in humans, by inexpensive methods with high throughput [27]. A
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great deal of knowledge on human amino acid metabolism has also been collected over the last
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three decades through the monitoring of plasma amino acid levels. Fisher’s ratio, the ratio
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between branched chain amino acids (L-leucine, L-valine, and L-isoleucine) and aromatic amino
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acids (L-phenylalanine and L-tyrosine), has historically been used as a marker of liver disorders 13
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[28, 29]. More recently, aminograms have been integrated to create a novel “Amino Index” that
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aids in the assessment of non-hepatic conditions [30, 31]. As an example, in lung cancer patients,
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two tissue-free amino acids (L-glutamate and glycine) were found to be increased in
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concentration, while the concentrations of two other amino acids (L-lysine and L-ornithine) were
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found to be significantly reduced [32]. For most neonatal mass-screenings, which test for
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genetic errors of amino acid metabolism, enzymatic methods are desirable. The Asano group
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developed a spectrophotometric recycling assay for the quantification of L-phenylalanine using
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PheDH [33] and a separate enzyme chip assay for the microquantification of L-phenylalanine
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[34] have been reported. Furthermore, directed mutagenesis of the enzyme, converting it into an
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NAD+-dependent L-methionine dehydrogenase, has been reported as a test for homocystinuria; a
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symptom of cystathionine β-synthase deficiency [35]. Investigators have identified various
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L-amino
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Fluorimetric assays utilizing Bacillus-derived NAD+-dependent LeuDH [36], or L-alanine
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dehydrogenase (EC 1.4.1.1) [37], were used to determine blood plasma levels of branched chain
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amino acids or L-alanine, respectively. An L-proline sensor was developed by linking an
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immobilized, polymerized mediator and thermostable dye to L-proline dehydrogenase (EC
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1.5.99.B2) [38]. The specific enzymatic determination of L-threonine was devised by using a
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newly discovered L-threonine 3-dehydrogenase (EC 1.1.1.103) from Cupriavidus necator
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NBRC 102504 [39]. Enzymatic assays using dehydrogenases have also been developed for
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D-amino
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molecular selection” using the L-amino acid auxotroph by some researchers, and these enzymes
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will be applied to determine L-amino acids for new diagnosis.
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acid dehydrogenases, which are used for determining the corresponding L-amino acids.
acids [40, 41]. The enzyme properties will be modified by “growth-dependent
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Acknowledgements 14
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This work was supported by JST ERATO Asano Active Enzyme Molecule Project
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(Grant Number JPMJER1102), Japan. This work was also supported by a Grant-in-Aid for
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Scientific Research (S) from the Japan Society for Promotion of Sciences (Grant Number
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17H06169) awarded to Y. Asano.
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dye-linked L-proline dehydrogenase and polymerized mediator, Sci. Technol. Adv. Mater., 7
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(2006) 243-248.
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necator and its use in determination of L-threonine, Anal Biochem, 410 (2011) 44-56.
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D-isoleucine
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Figure legends
427
Fig. 1 Growth-dependent molecular selection of a thermostable TrpDH variant [5]. (A) In vivo
428
selection following cultivation in minimum medium supplemented with IPA. (B) Growth curve
429
in modified M9 medium. E. coli ∆trpB harboring pUC19 (open circles), E. coli ∆trpB harboring
430
the wild type gene (open circles), and E. coli ∆trpB harboring a mutant gene (closed circles).
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Fig. 2 Thermal stabilities of TrpDH wild-type and mutant enzymes (placed on ice). Wild-type
433
enzyme (open circles), L59F/D168G/A234D/I296N variant enzyme (closed circles), L59F
434
variant enzyme (open triangles), D168G variant enzyme (closed triangles), A234D variant
435
enzyme (open squares), and I296N variant enzyme (closed squares).
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436 437
Fig. 3 L-Tryptophan quantification by TrpDH. (A) Schematic diagram of the TrpDH assay.
438
L-tryptophan
439
The reaction can be monitored at 340 nm [5]. Moreover, NADH thus generated is amplified by
440
the diaphorase cycling system to produce resazurin [35]. This reaction can be monitored at 550
441
nm. (B) Relationship between L-tryptophan concentration and absorbance value in MilliQ (open
442
circles) and human plasma (closed circles) [5]. The sample contains an endogenous
443
L-tryptophan,
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is oxidized by TrpDH in the presence of NAD+, which generates IPA and NADH.
and is spiked with various concentration of L-tryptophan.
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Table 1 Various enzyme screening by growth-dependent molecular evolution techniques Enzyme
Purpose
Host for screening
Screening strategy
References
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On minimal medium containing D-tryptophan, only the Broad
specificity
amino
acid
of
substrate
L-Tryptophan auxotroph E. coli
JM101: ∆tnaA, ∆trpABCDE
specificity
L-tryptophan auxotroph E. coli, harboring the mutant protein with increased tryptophan racemase activity, was
[7]
able to convert sufficient D-tryptophan to L-tryptophan
SC
racemase from P. putida IFO 12996
Modification
required for growth.
Threonine
aldolase
from
Caulobacter crescentus CB15
Increase
of
activity
of
L-threonine aldolase
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On medium containing β-hydroxy-α-amino acids, only the
Glycine auxotroph E. coli
strain harboring the protein with high activity, was able to convert sufficient β-hydroxy-α-amino acids to glycine
[8]
required for growth. On minimal medium containing DL-threo-β-phenylserine,
L-Tryptophan dehydrogenase from
N. punctiform ATCC29133
446
only the strain harboring the protein with L-threonine
threonine aldolase is delated: P.
aldolase
putida KT2440∆taPp
L-threo-β-phenylserine
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cerevisiae
Strain in which a gene encoding
aldolase
Increase
of
activity
of
dehydrogenase
stability
and
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xylosoxidans DSM2402, and S.
Identification of L-threonine
L-tryptophan
L-Tryptophan auxotroph E. coli
JM109: ∆trpB,
activity,
was
able
to
convert
sufficient
growth. On minimal medium containing indole-3-pyruvic acid, only the L-tryptophan auxotroph E. coli, harboring the mutant the protein with increased tryptophan racemase
acid to L-tryptophan required for growth.
22
[9]
to benzaldehyde required for
activity, was able to convert sufficient indole-3-pyruvic
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Threonine aldolases from E. coli, A.
[5]
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Method
Analysis time
Detection range
(min)
(µM)
Instrumental assay HPLC
30
0 - 1,000
Electrospray ionization tandem mass spectrometry
10
0.006 - 95
Flow-injection
amperometric
biosensor
based
Stability of enzyme
(Enzymes are not used in this assay.) (Enzymes are not used in this assay.)
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Enzymatic assay
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Table 2 Instrumental and enzymatic assays for L-tryptophan quantification
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447
Reference
[10] [11]
The biosensor was quite stable over a 6 month period
on
3
immobilized L-tryptophan 2-monooxygenase
100 - 50,000
when operated at room temperature with the biocatalyst
[12]
o
stored at 8 C. More than 90% activity of StaO was lost after 1 day of
Colorimetric assay, coupling L-tryptophan oxidase (StaO and
10 - 60
Monitoring of absorbance change at 340 nm by L-tryptophan
30
EP
dehydrogenase wild type
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VioA) and peroxidase
Monitoring of absorbance change at 340 nm by L-tryptophan
10 - 60
0 - 100
0 - 150
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dehydrogenase L59F/D168G/A234D/I296N variant enzyme
448
0 - 100
23
storage at 4oC, and no significant decrease in activity was observed after 1 week in 30% glycerol. 30% activity of
[13, 26]
VioA was obtained after 1 week of storage at 4oC More than 90% activity was lost after 1 day of storage at 4oC, and no significant decrease in activity was observed
[14]
after 3 days in 20% glycerol. 90% activity remained after 1 day of storage at 4oC.
[5]
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Highlights NAD+-dependent TrpDH was discovered in scytonemin biosynthesis of cyanobacteria.
3
Thermostable TrpDH variant was screened by growth-dependent molecular selection.
4
L-Trp
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quantification was performed using the TrpDH variant.
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1
S0003-2697(18)30995-3
DOI:
https://doi.org/10.1016/j.ab.2019.05.010
Reference:
YABIO 13321
To appear in:
Analytical Biochemistry
Received Date: 28 January 2019 Revised Date:
11 May 2019
Accepted Date: 13 May 2019
Please cite this article as: D. Matsui, Y. Asano, Creation of thermostable L-tryptophan dehydrogenase by protein engineering and its application for L-tryptophan quantification, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2019.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Running title: Protein engineering for L-tryptophan quantification
2 3
Subject Category: Enzymatic Assays and Analyses
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Creation of thermostable L-tryptophan dehydrogenase by protein engineering and its
6
application for L-tryptophan quantification
Daisuke Matsui1, 2 and Yasuhisa Asano1, 2†
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1
11
University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
12
2
13
939-0398, Japan
Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama
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Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural
15
†
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E-mail: [email protected]
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To whom correspondence should be addressed. Tel: +81-766-56-7500; Fax: +81-766-56-2498;
1
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List of abbreviations
18
HPLC
High performance liquid chromatography
19
IPA
Indole-3-pyruvic acid
20
LB
Luria-Bertani
21
LeuDH
L-Leucine
22
NAD+
Nicotinamide adenine dinucleotide
23
NADH
Nicotinamide adenine dinucleotide – hydrogen
24
NMR
Nuclear Magnetic Resonance
25
NpR1275
L-Tryptophan
26
PCR
Polymerase chain reaction
27
PheDH
L-Phenylalanine dehydrogenase
28
StaO and VioA
L-Tryptophan
29
taPp
Threonine aldolase gene
30
TMO
L-Tryptophan
31
tnaA
Tryptophanase gene
32
Tris
Tris(hydroxymethyl)aminomethane
33
trpABCDE
34
trpB
Tryptophan synthase beta chain gene
35
TrpDH
L-Tryptophan
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dehydrogenase gene
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oxidase
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2-monooxygenase
Component operon of tryptophan biosynthetic pathway
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dehydrogenase
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dehydrogenase
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37 38
Abstract L-Tryptophan
dehydrogenase is a new NAD+-dependent amino acid dehydrogenase
discovered in Nostoc punctiforme. The enzyme is involved in scytonemin biosynthesis and is
40
highly selective toward L-tryptophan. By a growth-dependent molecular evolution technique, a
41
thermostable mutant enzyme was selected successfully. L-Tryptophan concentration in human
42
plasma was successfully determined using the thermostable mutant of L-tryptophan
43
dehydrogenase.
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1. Introduction L-Tryptophan is
an essential amino acid originally isolated from enzymatic hydrolysis of
casein and the structure was identified by Hopkins and Cole in 1902 [1]. L-Tryptophan is also
47
known as an important amino acid in two biosynthetic pathways; first is the biosynthesis of
48
nicotinamide adenine dinucleotide via formylkynurenine and kynurenine, and the second is the
49
biosynthesis of melatonin via 5-hydroxytryptophan and serotonin. Recently, a significant
50
correlation between plasma tryptophan concentration and depressive illness was reported [2]. It
51
has been shown that L-tryptophan is linked to fibromyalgia [3], and L-tryptophan has recently
52
been identified as a useful biomarker for diagnosing inflammatory bowel disease [4]. Because
53
of these clinical implications, a simple assay method for quantification of L-tryptophan is
54
expected to greatly benefit the medical community.
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L-Tryptophan
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dehydrogenase (TrpDH, EC1.4.1.19) is useful for the determination of
56
L-tryptophan
57
throughput clinical use. Therefore, the Asano group attempted to improve the thermal stability
58
of TrpDH by utilizing new methods to develop a simple and rapid enzymatic method to
59
determine L-tryptophan concentration [5]. Directed evolution is a powerful tool used to identify
60
variants with desired properties such as high stability, but the process is problematic in that it
61
requires screening large libraries. In order to improve the hit-rate of beneficial variants, the
62
authors utilized directed evolution to obtain a mutant enzyme by the combination of random
63
mutagenesis of the gene using error-prone PCR and the transformation into an L-tryptophan
64
auxotroph, “growth-dependent molecular selection”.
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concentration but the enzyme is not stabile and therefore not suitable for high
In this review, we summarize how to reveal the characteristics of a new NAD+-dependent
66
TrpDH of the scytonemin biosynthesis pathway in cyanobacteria Nostoc punctiforme ATCC
67
29133, and how to utilize the protein engineering techniques to improve the properties of the 4
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TrpDH. Characteristics of TrpDH variant and determination of L-tryptophan in human plasma
69
using the variant are also described.
71
2. Materials and method
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74
2.1. Investigation of TrpDH from N. punctiform ATCC 29133
NpR1275 were amplified from N. punctiforme ATCC 29133 genomic DNA, and the
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tryptophan dehydrogenase activity of the recombinant enzyme was confirmed using a
76
spectrophotometic assay measuring the formation of NADH [6]. To investigate substrate
77
specificity, 1.0 mM each of twenty proteolytic amino acids and D-tryptophan were used as
78
substrates [5].
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2.2. Enzyme selections by the growth-dependent molecular evolution technique
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2.2.1. Selection of broad specificity amino acid racemase for modification of substrate
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specificity [7]
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Random mutagenesis on a gene of broad specificity, namely amino acid racemase from
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Pseudomonas putida IFO 12996, was carried out by error-prone PCR method, and Escherichia
85
coli JM101: ∆tnaA, ∆trpABCDE harboring the mutated gene were grown in M9 medium
86
containing 5 mg/L D-tryptophan and 50 mg/L casamino acids at 37°C for 16 h. The activities of
87
colony-forming mutants were compared by means of a colorimetric assay mixture which
88
consists 10 mM Tris-HCl (pH 8.5), 1 U/mL D-amino acid oxidase, 100 U/mL peroxidase, 5 mM
89
phenol and 1 mM 4-amino antipirine.
90
2.2.2. Selection of L-threonine aldolase for increase of activity [8]
91
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Cells containing a threonine aldolase gene library were grown in LB medium containing 150 5
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µg/mL ampicillin, and serial dilutions were plated in parallel onto minimal M9G plates lacking
93
glycine but containing 10 ng/mL or 50 ng/mL tetracycline and 0.2 mM L-threonine or 0.2 mM
94
L-allothreonine. The conversion
95
monitored by UV spectroscopy (∆ε279 nm=1400 M-1cm-1).
96
2.2.3. Identification of L-threonine aldolase [9]
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of L-β-threo-phenylserine to benzaldehyde and glycine was
P. putida KT2440 ∆taPp-carrying plasmids with threonine aldolase genes were cultivated overnight at 30 °C in 5 mL LB medium containing 50 µg/mL kanamycin, and a final cell
99
concentration corresponding to an optical density of 0.05 at 600 nm was used to inoculate 5-15
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mL of minimal medium M9 with different concentrations of DL-threo-β-phenylserine as sole
101
carbon source containing the corresponding antibiotic. The threonine aldolase activity towards
102
threonine was measured spectrophotometrically using UV absorbance at 340 nm by coupling
103
the reduction of the formed acetaldehyde with yeast alcohol dehydrogenase at 25°C.
104
2.2.4. Selection of L-tryptophan dehydrogenase for increase of stability and activity [5]
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E. coli ∆trpB was transformed with the pUC19 carrying random mutated TrpDH gene, and E. coli ∆trpB harboring the mutated gene were grown in 5.0 mL of a modified M9 medium
107
containing 5.0 mg/L indole-3-pyruvic acid, 100 µg/mL ampicillin, and 0.5 mM
108
isopropyl-β-D-thiogalactopyranoside at 30oC for 16 h. TrpDH activity of the crude extract was
109
determined by monitoring the reductionof β-NAD+ at 340 nm at 30◦C.
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2.3 Instrumental and enzymatic assays for L-tryptophan quantification
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2.3.1. High-performance liquid chromatography (HPLC) [10]
113
HPLC analysis was performed using a Waters 600E pump, gradient controller (Waters
114
Chromatography Corp., Milford, MA, USA), and 100 mm x 4.7 mm I.D., packed with
115
Partisphere 5 µm C18 (Whatman, Clifton, NJ, USA). Separations were achieved at ambient 6
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temperature by isocratic elution at a flow-rate of 1.0 mL/min, and the quantification were
117
carried out with a Waters 470 scanning fluorescence detector connected on line with a Waters
118
994 programmable photodiode array detector.
119
2.3.2. Electrospray ionization tandem mass spectrometry [11]
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Electrospray ionization tandem mass spectrometry analysis was carried out using an LC
121
system (LC Packings Ultimate capillary HPLC system, Amsterdam, The Netherlands), a 150 x
122
0.5 mm column packed with 3 µm C18 particles (ODS AQ, YMC), and a PE-Sciex API 365
123
triple quadrupole mass spectrometer (PE-Sciex, Concord, Canada) equipped with pneumatically
124
assisted electrospray ionization interface (Ionspray).
125
2.3.3. Flow-injection amperometric biosensor based on immobilized L-tryptophan
126
2-monooxygenase [12]
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Tryptophan-2-monooxygenase from P. savastanoi was produced in E. coli using a pUC19
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expression system, and tryptophan determination was carried out in continuously aerated 100
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mM Tris buffer, pH7.8, at a flow rate 3.5 mL/min, at 25°C using the amperometric batch unit
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with a Clark electrode and flow-injection amperometric system 'MultiFerm' (YerPhI, Armenia).
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2.3.4. Colorimetric assay, coupling L-tryptophan oxidases and peroxidase [13]
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Reaction mixtures contained 20 mM Tris-HCl (pH 9.0), 0-100 µM L-tryptophan, 1 mM
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4-aminoantipyrine,1 mM phenol, 15 U/mL horseradish peroxidase (Wako), and 10 mU/mL
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tryptophan oxidase solution in a final volume of 200 µL. Absorbance was measured at 505 nm
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by the microplate reader at the end of the reaction, using an extinction coefficient of 6.4 mM-1
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cm-1 for the quinoneimine dye product.
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2.3.5. Monitoring of absorbance change at 340 nm by L-tryptophan dehydrogenase [5, 14]
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The reaction was performed in a total volume of 200 µL containing100 mM glycine-KCl-KOH buffer (pH 11.5), 2.5 mM β-NAD+, and an appropriate amount of the sample. 7
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The reaction was started by the addition of enzyme, and was followed by the absorbance at 340
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nm using a microplate spectrophotometer at 30◦C for 10-30 min. The absorbance changes for
142
L-tryptophan
143
enzyme from the absorbance values with enzyme in each L-tryptophan concentration. In the
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assay of L-tryptophan in human plasma, human plasma was purchased from a commercial
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source (Cosmo Bio; Tokyo, Japan).
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concentration were determined by subtracting the absorbance values without
3. Results
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3.1. Screening of TrpDH in a new biosynthesis pathway and selection of a thermostable
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variant
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3.1.1. New screening of TrpDH from the scytonemin biosynthesis pathway Various plants such as pea, spinach, and tobacco contain active L-tryptophan
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dehydrogenases (EC 1.4.1.19), which is the primary enzyme of the indolylpyruvate pathway in
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plants [15]. Because these enzymes have not been purified and the genes not cloned,
155
characteristics such as substrate specificities have not been investigated.
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The indole-alkaloid scytonemin is the most common and widespread sunscreen among
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cyanobacteria; its biosynthetic route is shown in Scheme 1. In addition, it was revealed that an
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18-gene cluster (NpR1276 to NpR1259) [16] is responsible for scytonemin biosynthesis in the
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cyanobacterium N. punctiforme ATCC 29133 [17]. The scytonemin biosynthetic genes are
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highly conserved in other cyanobacteria [16]. Balskus and Walsh assigned the accepted
161
functions to each gene in the scytonemin gene cluster [18], and revealed that NpR1275
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resembled L-leucine dehydrogenase (LeuDH, EC 1.4.1.9, PDB code 1LEH) from B. sphaericus
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(sequence identity 46% with LeuDH). The overexpressed protein was shown to catalyze the
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oxidative deamination of L-tryptophan (referred to here as L-tryptophan dehydrogenase
165
(TrpDH)), as revealed by a spectrophotometric assay measuring the formation of NADH [6, 18]. In order to determine the substrate specificity of the protein encoded by NpR1275 in N.
167
punctiforme ATCC 29133, an expression plasmid containing the gene and expressed in E. coli
168
was constructed. The activity of the recombinant enzyme against 20 different of L-amino acids
169
and their derivatives was measured. The purified enzyme catalyzes oxidative deamination (1.03
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U/mg) only of L-tryptophan [14]. A homology model of TrpDH with the substrate L-tryptophan
171
using the structural information of LeuDH as a template was also constructed. The structure
172
folded into two domains (domain I and domain II) separated by a deep cleft, which is thought to
173
be the substrate binding site. The Ohshima group also cloned the homologous gene from N.
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punctiforme NIES-2108 and revealed the characteristics of its TrpDH [19]. NMR analysis of the
175
hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that
176
TrpDH has a pro-S (B-type) stereospecificity similar to that of the
177
glutamate/leucine/phenylalanine/valine dehydrogenase family. Furthermore, the characteristics
178
and thermal stability of TrpDH for application in an L-tryptophan assay was examined. TrpDH
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from N. punctiforme ATCC 29133 exhibits specific dehydrogenase activity for L-tryptophan and
180
shows a low Km value (0.201 mM). Once again, the enzyme is not stabile and therefore not
181
suitable for high throughput clinical use. [14]. Most of the reports on enzyme stabilization focus
182
on the effect of additives such as glycerol, sorbitol, and trehalose [20]. TrpDH also shows
183
activity following the addition of 20% glycerol, but other additives increase the absorbance
184
spontaneously in the L-tryptophan assay. Therefore, the Asano group attempted to stabilize the
185
enzyme by directed evolution [5].
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3.1.2. Selection of thermostable TrpDH by the growth-dependent molecular evolution
187
technique
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Directed enzyme evolution has been used in the past two decades as a powerful tool for generating enzymes with desired properties. Enzyme variants have evolved under extreme
190
conditions, such as in high temperatures, acidic and alkaline environments, and organic solvents,
191
[21, 22] resulting in their improved catalytic activity and specificity for new substrates [23, 24].
192
The typical library size needed for directed enzyme evolution is many orders of magnitude
193
larger than the number of protein variants that can be screened in a reasonable period. The
194
bottleneck for most directed enzyme evolution endeavors is the availability of a genuinely
195
high-throughput screen or the ability to select for target activity.
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Numerous investigators developed the selection strategy, and the “growth-dependent molecular selection” as described below (Table 1). These strategies have become breakthrough
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techniques. The Kino group used an L-tryptophan auxotroph to modify an amino acid, leading to
199
a racemase enzyme with high tryptophan racemization activity [7]. On minimal medium
200
containing D-tryptophan, only the L-tryptophan auxotroph E. coli, harboring the mutant bar
201
protein with increased tryptophan racemase activity, was able to convert sufficient D-tryptophan
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to L-tryptophan required for growth. The Hilvert group developed a new genetic selection
203
system for threonine aldolases, where retro-aldolase activity was directly linked to cellular
204
growth by simultaneous inactivation of four essential genes involved in E. coli glycine
205
biosynthesis [8]. This glycine auxotrophic strain could only grow in minimal medium
206
supplemented with glycine or by coupling with a glycine-liberating enzyme. Recently, the
207
Gröger group developed a highly efficient selection system for L-threonine aldolase using P.
208
putida KT2440 [9]. L-Threonine aldolase genes of E. coli and Saccharomyces cerevisiae and the
209
D-threonine aldolase
210
strain P. putida KT2440 ∆taPp, followed by cultivation on minimal medium supplemented with
211
DL-threo-β-phenylserine. The
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gene of Achromobacter xylosoxidans were introduced into the selection
results demonstrate that only the selection strains with 10
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plasmid-encoded L-threonine aldolases were able to grow on this racemic amino acid specific
213
medium. The Asano group applied the “growth-dependent molecular selection” to TrpDH mutant
215
screening [5, 25]. The mutant library was constructed using the error-prone PCR method and E.
216
coli was transformed with a disrupted L-tryptophan synthase gene (E. coli ∆trpB). In the
217
screening of TrpDH mutants, the complementation of L-tryptophan auxotroph was employed as
218
an indicator of increased TrpDH activity and stability. The L-tryptophan auxotroph E. coli ∆trpB,
219
harboring a mutated gene encoding a TrpDH variant with higher TrpDH activity, was able to
220
convert a large amount of indole-3-pyruvic acid (IPA) to L-tryptophan (Fig. 1A). TrpDH activity
221
of colonies cultured on M9 modified agar plates was measured. In these variants with higher
222
TrpDH activity, four amino acid substitutions (L59F, D168G, A234D, and I296N) were
223
identified. In order to compare the complementation of the L-tryptophan auxotroph, E. coli
224
∆trpB was transformed with plasmids containing either wild type or a variant gene insertion.
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Growth time of transformants harboring wild type and mutant genes were 0.16 h-1 and 0.27 h-1,
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respectively (Fig. 1B), which indicated that the improved properties of the mutant enzyme are
227
involved in the complementation of the L-tryptophan auxotroph.
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3.2. Characteristics of TrpDH and L-tryptophan quantification using various assay
230
3.2.1. Characteristics of TrpDH variants [5]
231
In the variants with higher TrpDH activities than wild type, the following amino acid
232
substitutions were identified: L59F/D168G/A234D/I296N (TrpDH-4mut). Single point
233
mutations at each amino acid residue were introduced into the wild type enzyme, and properties
234
of the mutant enzymes were compared with those of the wild type enzymes. When the activities
235
were measured after 72 hours and the residual activities were calculated, the Asp168 and Ile296 11
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substitutions increased the stability of the mutant (closed triangles and closed squares in Fig. 2),
237
while the enzymatic activity of TrpDH L59F and A234D increased approximately 1.77 and
238
6.32-fold (an open triangle and an open square at 0 hour in Fig. 2), respectively, compared to that
239
of the wild type TrpDH. It was revealed that the specific activity and stability of TrpDH-4mut
240
(closed circles in Fig. 2) were higher than those of the wild type enzyme (open circles in Fig. 2).
241
3.2.2. L-Tryptophan quantification using TrpDH-4mut [5]
TrpDH-4mut was used for the determination of L-tryptophan (Fig. 3A), yielding absorbance
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values that increased linearly with an increase in L-tryptophan concentration, producing a
244
standard curve that was linear (open circles in Fig. 3B). Furthermore, the 0 - 30 µmol of
245
L-tryptophan
246
absorbance change at 340 nm was measured at 30°C for 30 min. The resulting plot was also
247
linear (closed circles in Fig. 3B). The same concentration of L-tryptophan was also assayed by
248
ultra-performance liquid chromatography. L-Tryptophan determination using TrpDH was
249
achieved by stabilizing the enzyme using protein engineering as described in this paper.
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3.3. Instrumental and the other enzymatic assays for determining L- tryptophan
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in human plasma, was incubated with 1.0 unit of TrpDH at pH 11.5, and the
To date, various instrumental methods using high performance liquid chromatography
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(HPLC) [10] or electrospray ionization tandem mass spectrometry [11] have been developed to
254
determine L-tryptophan levels in plasma (Table 2). The sensitively of these detection systems is
255
high, but these methods require specialized systems and long run times. An amperometric
256
biosensor with L-tryptophan 2-monooxygenase (TMO, EC 1.13.12.3) [12] and colorimetric
257
assays using L-tryptophan oxidases (EC1.4.3.-) and peroxidase have been reported as enzymatic
258
methods to determine L-tryptophan [13, 26].
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260 261
4. Discussion Microbial NAD+-dependent TrpDH, which catalyzes the oxidative deamination and reductive amination between L-tryptophan and IPA, was found in the scytonemin biosynthetic
263
pathway of N. punctiforme ATCC29133 [6, 18]. This TrpDH exhibits high specificity toward
264
L-tryptophan,
265
the present study suggest that metabolism-oriented screening is a promising strategy for
266
obtaining enzymes with thermostability, which are often difficult to obtain by random screening.
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The “growth-dependent selection system” was adapted for the screening of thermostable
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but its instability is a drawback for L-tryptophan determination [14]. The results in
variants. In order to efficiently obtain higher-stability TrpDH variants from N. punctiforme
269
ATCC 29133 that would be useful in the determination of L-tryptophan, the Asano group
270
utilized the following approach: directed evolution, involving random mutagenesis of the
271
NpR1275 gene using error-prone PCR and its transformation into an L-tryptophan auxotroph, E.
272
coli ∆trpB. In this screening method, the transformants harboring the mutated NpR1275 genes
273
were cultivated in a medium containing the L-tryptophan precursor IPA as the main carbon
274
source [5]. This is because TrpDH is a key enzyme used by the E. coli transformants to
275
assimilate IPA. Using this selection method, one variant with high catalytic activity and stability
276
was obtained, and was used for the development of the L-tryptophan assay. This method is one
277
of only a few enzymatic L-tryptophan assays available.
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At the present time, developments in metabolomic approaches have enabled investigators to
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measure various metabolites in humans, by inexpensive methods with high throughput [27]. A
280
great deal of knowledge on human amino acid metabolism has also been collected over the last
281
three decades through the monitoring of plasma amino acid levels. Fisher’s ratio, the ratio
282
between branched chain amino acids (L-leucine, L-valine, and L-isoleucine) and aromatic amino
283
acids (L-phenylalanine and L-tyrosine), has historically been used as a marker of liver disorders 13
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[28, 29]. More recently, aminograms have been integrated to create a novel “Amino Index” that
285
aids in the assessment of non-hepatic conditions [30, 31]. As an example, in lung cancer patients,
286
two tissue-free amino acids (L-glutamate and glycine) were found to be increased in
287
concentration, while the concentrations of two other amino acids (L-lysine and L-ornithine) were
288
found to be significantly reduced [32]. For most neonatal mass-screenings, which test for
289
genetic errors of amino acid metabolism, enzymatic methods are desirable. The Asano group
290
developed a spectrophotometric recycling assay for the quantification of L-phenylalanine using
291
PheDH [33] and a separate enzyme chip assay for the microquantification of L-phenylalanine
292
[34] have been reported. Furthermore, directed mutagenesis of the enzyme, converting it into an
293
NAD+-dependent L-methionine dehydrogenase, has been reported as a test for homocystinuria; a
294
symptom of cystathionine β-synthase deficiency [35]. Investigators have identified various
295
L-amino
296
Fluorimetric assays utilizing Bacillus-derived NAD+-dependent LeuDH [36], or L-alanine
297
dehydrogenase (EC 1.4.1.1) [37], were used to determine blood plasma levels of branched chain
298
amino acids or L-alanine, respectively. An L-proline sensor was developed by linking an
299
immobilized, polymerized mediator and thermostable dye to L-proline dehydrogenase (EC
300
1.5.99.B2) [38]. The specific enzymatic determination of L-threonine was devised by using a
301
newly discovered L-threonine 3-dehydrogenase (EC 1.1.1.103) from Cupriavidus necator
302
NBRC 102504 [39]. Enzymatic assays using dehydrogenases have also been developed for
303
D-amino
304
molecular selection” using the L-amino acid auxotroph by some researchers, and these enzymes
305
will be applied to determine L-amino acids for new diagnosis.
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acid dehydrogenases, which are used for determining the corresponding L-amino acids.
acids [40, 41]. The enzyme properties will be modified by “growth-dependent
306 307
Acknowledgements 14
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This work was supported by JST ERATO Asano Active Enzyme Molecule Project
309
(Grant Number JPMJER1102), Japan. This work was also supported by a Grant-in-Aid for
310
Scientific Research (S) from the Japan Society for Promotion of Sciences (Grant Number
311
17H06169) awarded to Y. Asano.
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[41] Y. Tani, Y. Itoyama, K. Nishi, C. Wada, Y. Shoda, T. Satomura, H. Sakuraba, T. Ohshima, Y.
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Hayashi, T. Yabutani, J. Motonaka, An amperometric D-amino acid biosensor prepared with a
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thermostable D-proline dehydrogenase and a carbon nanotube-ionic liquid gel, Anal. Sci., 25
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(2009) 919-923.
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using an artificially created D-amino acid dehydrogenase, Biotechnology letters, 36
AC C
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Figure legends
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Fig. 1 Growth-dependent molecular selection of a thermostable TrpDH variant [5]. (A) In vivo
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selection following cultivation in minimum medium supplemented with IPA. (B) Growth curve
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in modified M9 medium. E. coli ∆trpB harboring pUC19 (open circles), E. coli ∆trpB harboring
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the wild type gene (open circles), and E. coli ∆trpB harboring a mutant gene (closed circles).
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Fig. 2 Thermal stabilities of TrpDH wild-type and mutant enzymes (placed on ice). Wild-type
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enzyme (open circles), L59F/D168G/A234D/I296N variant enzyme (closed circles), L59F
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variant enzyme (open triangles), D168G variant enzyme (closed triangles), A234D variant
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enzyme (open squares), and I296N variant enzyme (closed squares).
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Fig. 3 L-Tryptophan quantification by TrpDH. (A) Schematic diagram of the TrpDH assay.
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L-tryptophan
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The reaction can be monitored at 340 nm [5]. Moreover, NADH thus generated is amplified by
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the diaphorase cycling system to produce resazurin [35]. This reaction can be monitored at 550
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nm. (B) Relationship between L-tryptophan concentration and absorbance value in MilliQ (open
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circles) and human plasma (closed circles) [5]. The sample contains an endogenous
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L-tryptophan,
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is oxidized by TrpDH in the presence of NAD+, which generates IPA and NADH.
and is spiked with various concentration of L-tryptophan.
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Table 1 Various enzyme screening by growth-dependent molecular evolution techniques Enzyme
Purpose
Host for screening
Screening strategy
References
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On minimal medium containing D-tryptophan, only the Broad
specificity
amino
acid
of
substrate
L-Tryptophan auxotroph E. coli
JM101: ∆tnaA, ∆trpABCDE
specificity
L-tryptophan auxotroph E. coli, harboring the mutant protein with increased tryptophan racemase activity, was
[7]
able to convert sufficient D-tryptophan to L-tryptophan
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racemase from P. putida IFO 12996
Modification
required for growth.
Threonine
aldolase
from
Caulobacter crescentus CB15
Increase
of
activity
of
L-threonine aldolase
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Glycine auxotroph E. coli
strain harboring the protein with high activity, was able to convert sufficient β-hydroxy-α-amino acids to glycine
[8]
required for growth. On minimal medium containing DL-threo-β-phenylserine,
L-Tryptophan dehydrogenase from
N. punctiform ATCC29133
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only the strain harboring the protein with L-threonine
threonine aldolase is delated: P.
aldolase
putida KT2440∆taPp
L-threo-β-phenylserine
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cerevisiae
Strain in which a gene encoding
aldolase
Increase
of
activity
of
dehydrogenase
stability
and
EP
xylosoxidans DSM2402, and S.
Identification of L-threonine
L-tryptophan
L-Tryptophan auxotroph E. coli
JM109: ∆trpB,
activity,
was
able
to
convert
sufficient
growth. On minimal medium containing indole-3-pyruvic acid, only the L-tryptophan auxotroph E. coli, harboring the mutant the protein with increased tryptophan racemase
acid to L-tryptophan required for growth.
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[9]
to benzaldehyde required for
activity, was able to convert sufficient indole-3-pyruvic
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Threonine aldolases from E. coli, A.
[5]
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Method
Analysis time
Detection range
(min)
(µM)
Instrumental assay HPLC
30
0 - 1,000
Electrospray ionization tandem mass spectrometry
10
0.006 - 95
Flow-injection
amperometric
biosensor
based
Stability of enzyme
(Enzymes are not used in this assay.) (Enzymes are not used in this assay.)
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Table 2 Instrumental and enzymatic assays for L-tryptophan quantification
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Reference
[10] [11]
The biosensor was quite stable over a 6 month period
on
3
immobilized L-tryptophan 2-monooxygenase
100 - 50,000
when operated at room temperature with the biocatalyst
[12]
o
stored at 8 C. More than 90% activity of StaO was lost after 1 day of
Colorimetric assay, coupling L-tryptophan oxidase (StaO and
10 - 60
Monitoring of absorbance change at 340 nm by L-tryptophan
30
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dehydrogenase wild type
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VioA) and peroxidase
Monitoring of absorbance change at 340 nm by L-tryptophan
10 - 60
0 - 100
0 - 150
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dehydrogenase L59F/D168G/A234D/I296N variant enzyme
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0 - 100
23
storage at 4oC, and no significant decrease in activity was observed after 1 week in 30% glycerol. 30% activity of
[13, 26]
VioA was obtained after 1 week of storage at 4oC More than 90% activity was lost after 1 day of storage at 4oC, and no significant decrease in activity was observed
[14]
after 3 days in 20% glycerol. 90% activity remained after 1 day of storage at 4oC.
[5]
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Highlights NAD+-dependent TrpDH was discovered in scytonemin biosynthesis of cyanobacteria.
3
Thermostable TrpDH variant was screened by growth-dependent molecular selection.
4
L-Trp
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quantification was performed using the TrpDH variant.
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1