Beauveria bassiana ATCC 7159 contains an L-specific α-amino acid benzamidase

Article abstract of DOI:10.1007/s007060070095

Biotransformation of a series of racemic N-benzoyl α-amino acids by the fungus Beauveria bassiana ATCC 7159 results in isolation of the corresponding D-amino acid benzamides in high enantiomeric purity and yield.

Full text of DOI:10.1007/s007060070095

Monatshefte fuÈr Chemie 131, 667±672 (2000)
Beauveria bassiana ATCC 7159 Contains
an L-Speci®c a-Amino Acid Benzamidase
Ã
Aaron van Vliet, Nancy J. Ihasz, and Frances M. Brown
Herbert L. Holland , Peter R. Andreana, Reza Salehzadeh-Asl,
Institute of Molecular Catalysis, Department of Chemistry, Brock University, St. Catharines, ON
L2S 3A1, Canada
Summary. Biotransformation of a series of racemic N-benzoyl ꢀ-amino acids by the fungus
Beauveria bassiana ATCC 7159 results in isolation of the corresponding D-amino acid benzamides
in high enantiomeric purity and yield.
Keywords. Amino acid; Beauveria bassiana; Biocatalysis; Biotransformation, Kinetic resolution
Introduction
Enzymatic methods for the kinetic resolution of racemic ꢀ-amino acids are well
established [1]. Methods have been developed that involve the stereoselective
hydrolysis of carboxylic acid ester [2] or amino amide [3] derivatives; either D- or
L-speci®c hydrolysis can occur, and the product may be isolated as the free amino
acid or as the unhydrolyzed substrate [4]. These transformations are summarized in
Fig. 1.
Enantioselective hydrolyses involving amide derivatives of the amino group are
most frequently reported using an amino acylase enzyme preparation [5] or a whole
cell biocatalyst containing such an enzyme [6] for hydrolysis of the acetamide group.
Reports of the kinetic resolution of other amino amide derivatives of ꢀ-amino acids
by aminoacylase enzymes include inter alia examples of hydrolyses of N-chloro-
acetyl [7], N-tri¯uoroacetyl [7], N-methoxyacetyl [7], N-benzyl [8], and N-benzyl-
oxycarbonyl [8] derivatives.
The enzymatic hydrolysis of N-benzoyl (benzamide) derivatives of ꢀ-amino
acids has been only rarely reported, in spite of an early account of the use of
takadiastase for the enantioselective hydrolysis of such compounds [9]. A ``D-
deacylase powder'' from an unidenti®ed pseudomonad has been reported to
hydrolyze the D enantiomer of N-benzoylphenylalanine in an enantioselective
manner [10], and L-N-benzoylglutamic acid is converted to the free amino acid by an
enzyme described as urethane hydrolase IV from an unidenti®ed microbial source
[11], but the hydrolysis of N-benzoyl derivatives has not been systematically
Ã
Corresponding author

668
H. L. Holland et al.
Fig. 1. Kinetic resolution of amino acid derivatives
investigated as a means for kinetic resolution of the naturally occurring ꢀ-amino
acids.
The fungus Beauveria bassiana ATCC 7159 is one of the most frequently used
whole cell biocatalysts, being exceeded in application only by baker's yeast,
Pseudomonas putida, and Aspergillus niger [12]. It catalyzes a wide variety of
reactions which have recently been reviewed [13], including the hydrolysis of
amides. In this report we describe the use of B. bassiana for the L-speci®c enantio-
selective hydrolysis of a series of N-benzoyl ꢀ-amino acids.
Results and Discussion
Biotransformation of the N-benzoyl derivatives of racemic ꢀ-amino acids by
Beauveria bassiana ATCC 7159 results in the enantioselective hydrolysis of the
natural L-isomer, leaving the residual D-N-benzoyl amino acid in high enantio-
meric excess. These results are summarized in Fig. 2 and Table 1.
The enantiomeric excess (ee) of the products listed in Table 2 were determined
by chiral stationary phase HPLC analysis with reference to authentic enantiopure
standards. Yields refer to isolated, puri®ed material and are based on the total
amount of racemic substrate used. Low yields in the case of the sulfur-containing
amino acids may be attributable to the formation of the corresponding sulfoxides as
side products. Beauveria bassiana is known to convert N-protected thia-amino
acids to the corresponding sulfoxides with high diastereomeric selectivity [14], but
this reaction is slower than hydrolysis of the benzamide group, taking 3±5 days for
completion compared with the 1±2 days required for the hydrolytic process. A time
course study for the hydrolysis of DL-N-benzoylalanine is presented in Fig. 3, from
which it is apparent that complete conversion of the L-enantiomer has been
Fig. 2. Kinetic resolution of N-benzoyl amino acid derivatives by B. bassiana ATCC 7159

L-Speci®c ꢀ-Amino Acid Benzamidase from B. bassiana
669
Table 1. Kinetic resolution of N-benzoyl amino acid derivatives by B. bassiana ATCC 7159
(Æ)-N-Benzoyl ꢀ-amino acid substrate
Substituent
D-N-Benzoyl ꢀ-amino acid product
Yield/%
ee/%
Alanine
CH3
20
28
23
17
16
22
25
23
18
30
27
> 95
80
Asparagine
Aspartic acid
Cysteine
CH2CONH2
CH2CO2H
> 95
> 95
> 95
> 95
20
CH2SH
Ethionine
CH2CH2SCH2CH3
CH2CH2CO2H
CH2CH2CONH2
CH2CH(CH3)2
CH2CH2SCH3
CH2C6H5
Glutamic acid
Glutamine
Leucine
> 95
> 95
> 95
> 95
Methionine
Phenylalanine
Phenylglycine
C6H5
Pipecolinic acid
20
> 95
Proline
20
32
21
> 95
> 95
> 95
S-Methylcysteine
Tryptophan
CH2SCH3
Tyrosine
17
28
20
Valine
(CH3)2CH
> 95
a
Refer to structures of Fig. 2; complete structures are shown for pipecolinic acid and proline only
attained after 36±48 hours incubation. Similar studies were not performed for the
other substrates listed in Table 2, but on the basis of the data presented in Table 2 a
72 hour incubation period was selected as standard. The moderate ee observed for
hydrolyses of racemic N-benzoyl asparagine, glutamine and tyrosine may be
attributable to incomplete conversion of these substrates over the period of their
biotransformation.
The hydrolysis of benzamides by B. bassiana appears to be selective for ꢀ-amino
acid derivatives. Benzamides of ꢁ-amino acids such as 3-aminobutyric acid and 3-
amino-3-phenylpropionic acid were not hydrolyzed by B. bassiana, substrates being
recovered unchanged following incubation for ®ve days. In other studies [15±19]
many different amino substrates protected as their N-benzoyl derivatives have been
hydroxylated by B. bassiana without loss of the benzoyl group; indeed, presence of
the latter functionality in the substrate is often bene®cial to the yield and stereo-
selectivity of such processes, as the benzamide unit may act as a binding group for
the hydroxylase enzymes present in B. bassiana [18, 19]. As shown in the present

670
H. L. Holland et al.
Fig. 3. Time course for the hydrolysis of DL-N-benzoylalanine by B. bassiana ^: L-N-benzoyl
alanine, &: D-N-benzoyl alanine
study, however, the hydrolysis of racemic N-benzoyl ꢀ-amino acids by B. bassiana
presents a simple method for the preparation of the D-benzoyl ꢀ-amino acids listed
in Table 1 in moderate yield and, for most examples, high enantiomeric purity.
Experimental
Materials and methods
1
Melting points were determined on a Ko¯er hot stage and are uncorrected. The H NMR spectra
were recorded on a Bruker Avance series 300 spectrometer in CD3OD solution using residual
CH3OH as the internal standard; chemical shifts are reported in ppm (ꢂ), and the multiplicities are
quoted as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). The 13C NMR spectra were
recorded at 75 MHz on the same spectrometer in CD3OD solution. Mass spectra were obtained in the
‡FAB mode using a Kratos 1S spectrometer. IR spectra were obtained using a Mattson Research
Series FTIR spectrometer. Optical rotations were recorded at ambient temperature in methanol as
solvent using a Rudolph Autopol 3 polarimeter. Enantiomeric excess (ee) was determined using an
ASTEC Chirobiotic TTM column 250Â4.6 mm with acetonitrile/methanol/acetic acid/triethylamine
455/545/2/2 solvent at a ¯ow rate of 1 cm3/min with UV detection at 254 nm. TLC was performed
on Merck silica gel F254 plates (0.2 mm), and column chromatography used Merck silica gel 9385
(230±400 mesh). Beauveria bassiana ATCC 7159 was maintained on Sabouraud dextrose agar
slopes, grown at 26ꢀC and stored at 4ꢀC.
Preparation of substrates
With the exception of the N-benzoyl tyrosine, all substrates were prepared in 85±95% yield by the
following general procedure: the racemic amino acid (1 g) was dissolved in sodium hydroxide
solution (20 cm3, 2 M), and the solution cooled to 0ꢀC. Benzoyl chloride (1.1 mole equivalents) was
added in ten portions over a period of 1 h while the solution was stirred vigorously. The pH was
checked periodically and adjusted if necessary to >12 by the addition of 2 M NaOH. When the
addition was complete, the solution was stirred at room temperature for 2 h, then re-cooled to 0ꢀC
and adjusted to pH ˆ 2 by the addition of 5% HCl. The precipitated product was separated by

L-Speci®c ꢀ-Amino Acid Benzamidase from B. bassiana
671
®ltration, dried, and crystallized from 95% ethanol. Products from proline and pipecolinic acid did
not precipitate in crystalline form and were isolated by extraction with CH2Cl2 and evaporation of
the extract to give a solid residue which was then crystallized. All products gave satisfactory
spectroscopic data and melting points in agreement with those reported [20].
N-Benzoyltyrosine was prepared by a modi®cation of the above procedure in which methanol
(10 cm3) was added to the reaction mixture following the 2 h stirring period at room temperature.
The mixture was then stirred at room temperature overnight and subsequent steps performed as
described above.
Biotransformations with B. bassiana
A growth medium (1 dm3) composed of glucose (10 g) and corn steep liquor (20 g) in distilled water,
adjusted to pH 4.85 with 1 M NaOH, was distributed in 5 1 dm3 Erlenmeyer ¯asks which were
stoppered with foam plugs and sterilized by autoclaving at 121ꢀC for 20 min. The ¯asks were
allowed to cool and then inoculated under sterile conditions with B. bassiana taken from a 3-day old
agar slope. The ¯asks were allowed to stand overnight at 27ꢀC and then placed on a rotary shaker
(100orbit) at 180 rpm, 27ꢀC. After three days, a solution of the appropriate substrate (0.5 g) in 95%
ethanol (10 cm3) was added, and growth was allowed to continue for a further 3 days. The fungal
mass was removed by ®ltration, and the ®ltrate was adjusted to pH 3 and continuously extracted with
CH2Cl2 for 4 days. The extract was then evaporated to give a residue that was examined by TLC and
chiral stationary phase HPLC and subjected to column chromatography using ethyl acetate/methanol
solvent mixtures containing 2% acetic acid. The solvent composition was changed from 0%
methanol to 40% methanol in 10% steps, and elution with the ®nal mixture was continued until all
product was removed from the column. All products had physical constants (melting points and
optical rotation data) as reported [20] and exhibited spectroscopic data consistent with their
structures.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of Canada
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Received December 20, 1999. Accepted January 21, 2000