RSC Advances
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COMMUNICATION
DOI: 10.1039/C4RA13646B
Notes and references
a
Institute of Bioengineering, Zhejiang University of Technology,
Hangzhou, Zhejiang 310014, People’s Republic of China. Tel: +86ꢀ571ꢀ
88320614; fax: +86ꢀ571ꢀ88320630; eꢀmail: zhengyg@zjut.edu.cn.
b Engineering Research Center of Bioconversion and Biopurification of
the Ministry of Education, Hangzhou, Zhejiang 310014.
†Electronic Supplementary Information (ESI) available: [Experimental
and 1H and 13C NMR spectra]. See DOI: 10.1039/c000000x/
1. G. DeSantis, K. Wong, B. Farwell, K. Chatman, Z. L. Zhu, G.
Tomlinson, H. J. Huang, X. Q. Tan, L. Bibbs, P. Chen, K.
Kretz and M. J. Burk, J Am Chem Soc, 2003, 125, 11476ꢀ
11477.
Fig 3. The process of the formation of HN by biotransformation of
(S)ꢀCHBE at 200 g⋅Lꢀ1 using the whole cells of HHDHꢀPL. Symbols:
concentration of (S)ꢀCHBE (□), concentration of HN (○), and ee of
(R)ꢀHN (■).
2. S. K. Ma, J. Gruber, C. Davis, L. Newman, D. Gray, A. Wang, J.
Grate, G. W. Huisman and R. A. Sheldon, Green Chem, 2010,
12, 81ꢀ86.
3. J. E. T. V. Vlieg, L. X. Tang, J. H. L. Spelberg, T. Smilda, G. J.
Poelarends, T. Bosma, A. E. J. van Merode, M. W. Fraaije and
D. B. Janssen, J Bacteriol, 2001, 183, 5058ꢀ5066.
At last we scaled up the process by biotransformation of 200 g⋅Lꢀ
1 of (S)ꢀCHBE under the optimal conditions (40 oC and pH 8.0) (Fig.
3). The reaction was carried out in a 1 L jacket threeꢀneck bottom
containing 400 mL 100 mM PBS buffer (pH 7.5). To it 50% sulfuric
acid was added to adjust pH to 4.0 and then 30% of (wt/vol) sodium
cyanide solution was added to pH 7.5. The mixture temperature was
4. R. J. Fox, S. C. Davis, E. C. Mundorff, L. M. Newman, V. Gavrilovic,
S. K. Ma, L. M. Chung, C. Ching, S. Tam, S. Muley, J. Grate,
J. Gruber, J. C. Whitman, R. A. Sheldon and G. W. Huisman,
Nat Biotechnol, 2007, 25, 338ꢀ344.
o
heated to 40 C and followed by adding 20 g lyophilized cells of
HHDHꢀPL and 100 g (S)ꢀCHBE (0.6 mol). Subsequently, a pH stat
was used to control the pH at 8.0±0.05 by adding 30% of sodium
cyanide solution. The reaction process was monitored by taking
samples from the mixture for GC analysis. The reaction was
completed after 14 h when the conversion of (S)ꢀCHBE reached 95%.
To the mixture 8 g CaCl2 was added to stir for 1 h and the cells were
removed through filtration. The separated mixture was extracted
three times with ethyl acetate (300 mL). The organic extracts were
combined and dried over anhydrous sodium sulphate. The ethyl
acetate was removed by evaporation under vacuum and remained
80.68 g (0.513 mol and yield 85%) yellow liquid.
In conclusion, a new HHDH gene HHDHꢀPL was
identified and heterologously overexpressed in E. coli in this
study. The sequence alignment assay and substrate specificity
study indicated that HHDHꢀPL possessed different properties
from the previously characterized HHDHs. In addition, HHDHꢀ
5. G. HasnaouiꢀDijoux, M. M. Elenkov, J. H. L. Spelberg, B. Hauer and
D. B. Janssen, Chembiochem, 2008,
6. R. M. Haak, C. Tarabiono, D. B. Janssen, A. J. Minnaard, J. G. de
Vries and B. L. Feringa, Org Biomol Chem, 2007, , 318ꢀ323.
7. M. M. Elenkov, I. Primozic, T. Hrenar, A. Smolko, I. Dokli, B.
9, 1048ꢀ1051.
5
SalopekꢀSondi and L. X. Tang, Org Biomol Chem, 2012, 10
,
5063ꢀ5072.
8. L. S. CampbellꢀVerduyn, W. Szymanski, C. P. Postema, R. A.
Dierckx, P. H. Elsinga, D. B. Janssen and B. L. Feringa, Chem
Commun, 2010, 46, 898ꢀ900.
9. M. M. Elenkov, H. W. Hoeffken, L. Tang, B. Hauer and D. B.
Janssen, Adv Synth Catal, 2007, 349, 2279ꢀ2285.
10. F. Xue, Z. Q. Liu, N. W. Wan and Y. G. Zheng, Appl Biochem
Biotechnol, 2014, 174, 352ꢀ364.
PL was applied in biotransformation of (
S)ꢀCHBE at a
11. M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna, P. A.
McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A.
Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and D. G.
Higgins, Bioinformatics, 2007, 23, 2947ꢀ2948.
concentration of 200 g
⋅
Lꢀ1, which generate 95% conversion and
85% yield. To the best of our knowledge, this was the first
report to prepare HN by using a natural HHDH at a high
substrate concentration. The high substrate tolerance to (S)ꢀ
CHBE suggested HHDHꢀPL was a promising biocatalyst for
commercially preparation of HN, as well as for academic
research. However, the substrateꢀtoꢀcatalyst ration was high and
the catalytic efficiency was inferior to the method reported by
Steven K. Ma.2 The large amount of biocatalysts in the process
will have an effect on both cost and the product separation.
Hence, in our future work we will try to engineer the catalytic
activity, stability and enantioselectivity of HHDHꢀPL for its
broader practical application including preparation of HN and
the other optically active secondary alcohols and epoxides.
This work was financially supported by 973 Program (No.
2011CB710806), National High Technology Research and
Development Program of China (No. 2012AA022201B) and Natural
Science Foundation of Zhejiang Province of China (No. Z4080032
and R311055).
12. Z. Y. You, Z. Q. Liu and Y. G. Zheng, Appl Microbiol Biotechnol
2013, 97, 9ꢀ21.
13. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D.
S. Goodsell and A. J. Olson, J Comput Chem, 2009, 30, 2785ꢀ
2791.
14. R. M. de Jong, J. J. W. Tiesinga, H. J. Rozeboom, K. H. Kalk, L.
Tang, D. B. Janssen and B. W. Dijkstra, Embo Journal, 2003,
22, 4933ꢀ4944.
4 | J. Name., 2012, 00, 1-3
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