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Discovery of a new a Fe-type nitrile hydratase efficiently hydrating
aliphatic and aromatic nitriles by genome mining
The name(s) and affiliation(s) of the author(s):
Xiaolin Pei1,2, Lirong Yang1, Gang Xu1, Qiuyan Wang2, Jianping Wu1
1 Institute of Bioengineering, Department of Chemical and Biological Engineering,
Zhejiang University, Hangzhou, 310028, PR China
2 Center for Biomedicine and Health, College of Life and Environmental Sciences,
Hangzhou Normal University, Hangzhou 310012, PR China
Correspondence:
Jianping Wu ([email protected]), Institute of Bioengineering, Department of Chemical
and Biological Engineering, Zhejiang University, Hangzhou, 310028, PR China. Tel.:
0086-571-87952363; Fax: 0086-571-87952009.
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1. Materials and Methods
1.1 Purification of the recombinant NHase_F1
For purification of the recombinant NHase_F1, the obtained supernatant was loaded
to a nickel chelate affinity column (Ni-NTA Resin, Bio Basic INC.) using loading
buffer (20 mM phosphate buffer, pH 7.5, 500 mM NaCl, and 50 mM imidazote).
Target NHase protein was recovered using elution buffer (20 mM phosphate buffer,
pH 7.5, 500 mM NaCl, and 250 mM imidazote), then desalted by HiTrap desalting
column (Amersham Biosciences).
1.2 Identification of nicotinamide
Identification of the reaction product was performed through the 1H NMR and mass
spectra. The 1H nuclear magnetic resonance (NMR, dimethylsulfoxide, 400 MHz) and
mass spectra were recorded with FX-90Q (Jeol, Japan) and LCMS-2010 (Shimadzu,
Japan) respectively.
1.3 Molecular modeling
The homology modeling of NHases from Pseudomonas putida F1 and Pseudomonas
chlororaphis B23 was conducted using Accelry Discovery Studio 3.0. The crystal
structure 3A8O, NHase from Rhodococcus erythropolis N771, was used as the model
structure. The obtained 3D structures were energy minimized using Smart Minimizer
algorithm with Max steps of 200 and RMS gradient of 0.1. The 3D structural
similarity of NHases from P. putida F1 and P. chlororaphis B23 were aligned using
Align Structures (3DMA program) with RMSD Cutoff of 2.5.
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Select Conserved motifs/Blocks as Probes
Sequence Analysis and Target Selection
Sequence Library Assembly
Select target phenotype NHase protein sequencesAlign protein sequences by ClustalW2Conserved protein sequences motifs/blocks
Genome databasesGenBank
Non-redundant (nr)Search tools
BLAST
Extract and select protein sequences that contain the conserved motif
Sequence analysisAlign sequences
PhylogenyDistance
Select at least one of the gene sequences
Design One or More Degenerate Primers to Target the Selected Gene Sequences
Cloning, Expression and Characterization
Fig. S1. Methodology for Fe-type NHases by genome mining.
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Fig. S2. (A) Multiple sequence alignment of putative active site from different
NHases. (B) The crystal structure of NHase and putative active site.
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Fig. S3. SDS-PAGE of purified recombinant NHase_F1 fractions. M: molecular
weight standard, lane 1: whole cell protein, lane 2: crude cell extract, lane 3 and 4:
Ni-NTA affinity chromatography and desalting
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9.0
25
8.6
98 8
.687
8.2
11 8
.191
8.1
68
7.6
15
7.5
06 7
.493
7.4
86 7
.474
1.00 0.99
2.04
0.98
1.07
9.0 8.5 8.0 7.5 7.0 PPM
Fig. S4. 1H NMR (dimethylsulfoxide [DMSO], 400 MHz) spectrum of nicotinamide
from 3-cyanopridine catalyzed by recombinant NHase_F1. Nicotinamide was
dissolved in DMSO-D6.
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Fig. S5. ESI-(+)-LC/MS spectrum of nicotinamide from 3-cyanopridine catalyzed by
recombinant NHase_F1. The mass peak with m/z 123.12 corresponds to the [M + H]+
of nicotinamide.
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