Endophytic fungi from Humiria balsamifera vegetal specie active as

Endophytic fungi from Humiria balsamifera vegetal
specie active as biocatalyst in transamination reaction
Larissa Zambe Pinheiro1*, Maria Sandra Ramos Queiroz2, Michelle Frazão
Muzitano2, Rodrigo Octavio Mendonça Alves de Souza3, Denise Oliveira
Guimarães2 and Ivana Correa Ramos Leal1.
1 Laboratório de Produtos Naturais e Ensaios Biológicos (LaProNEB)- Universidade Federal do Rio de Janeiro (UFRJ-RJ)
2 Laboratório de Produtos Naturais (Pólo Novo Cavaleiros/IMMT) - Universidade Federal do Rio de Janeiro (UFRJ-Macaé)
3 Lab. de Biocatálise e Síntese Orgânica- BOSS Group- Instituto de Química Universidade Federal do Rio de Janeiro (UFRJ-RJ)
*
*[email protected]
Keywords: biotransformation, chiral amines, endophytic fungi, Humiria balsamifera, transamination.
INTRODUCTION
The northern region of the Rio de Janeiro State is
recognized by its faunal and floristic richness since its
environmental characteristics favor the establishment of
continental biotas. The species Humiria balsamifera
(Humiriaceae) is part of this flora (Jurubatiba shoal) and
has never been studied by its endophytic fungi profile.
The endophytic micro-organisms are those associated
with the host healthy plant tissues and can provide
significant contribution as biotransformation agents. The
participation of micro-organisms associated with plant
species for biotechnological proposes, such as for
transamination reactions, is still scarce. This green
alternative may lead to favorable results compared to
the organic synthesis, allowing the production of quiral
amine blocks of great interest for the pharmaceutical
industries in mild conditions. Thus, this study aims to
promote the screening of endophytic fungi associated
with the vegetal species H. balsamifera as potential
biocatalysts for transamination reactions.
RESULTS AND DISCUSSION
The endophytic fungi isolated from H. balsamifera
vegetal species was coded as HB4a (Figure 1).
Figure 1: Endophytic fungi
HB4a isolated from Humiria
balsamifera
The HB4a fungus was investigated for its biocatalyst
capacity for producing acetophenone from racphenylethylamine. The conversion for the desired
product was investigated by spectrophotometer at 245
nm and GC-MS analyses (scheme 1).
Pre-fermentative
culture
medium
(potato
dextrose)
with3 plugs of HB4a. Rotation
at 120 rpm, 30°C and for five
Czapek Culture medium with
mycelial mass of the HB4a
and substrate. Rotation at
120 rpm,, 30 ° C for five days.
245
Partition with
ethyl acetate
Scheme 1: Culture of HB4a fluid and, in sequence, the
product analysis by spectrophotometer and GC-MS. The ωtransaminase enzyme was used as positive control.
A
B
Figure 2: Chromatogram obtained by GC-MS: (A) Culture
fluid with rac-phenylethylamine before reaction (negative
control) and (B) Culture fluid with rac-phenylethylamine after
forty-eight hours of reaction.
From the data obtained based on the calibration
curve of acetophenone by spectrophotometer and, by
GC-MS data, it was possible to observe that the
endophytic fungi HB4a showed 17% conversion into the
desired product (acetophenone). The optimized
conditions will be investigated by applying an
experimental design considering the variables:
temperature, stirring, pH and sacarose conc. It is also
noteworthy that the enantiomeric excess will be further
calculated by chiral HPLC analysis.
Based on this result, other racemic mixtures will be
tested
such
as
rac-butylamine,
racmethoxybenzylamide and rac- 1-Cyclohexylethylamine
CONCLUSION
Our data suggest that the fungi selected HB4a is
promising for the production of enzymes with
transaminase activity, justifying the proposal presented
here and the importance of continuing this investigation.
ACKNOWLEDGEMENTS
REFERENCES
JANECZKO, T.; DMOCHOWSKA-GLADYSZ, J.; KOSTRZEWASUSLOW, E. (2010) World J Microbiol Biotechnol, 26:2047–2051.
SCHATZLE, S.; HOHNE, M.; REDESTAD, E.; ROBINS, K.;
BORNSCHEUER, U. T. (2009) Anal. Chemical, 81, 8244–8248.
VII Workshop on Biocatalysis and Biotransformations and 1o Simposio Latinoamericano de Biocatalisis y Biotransformaciones