Acid phosphatases from Shigella flexneri (PhoN-Sf) [605] and Salmonella
enterica (PhoN-Se) [606] phosphorylate not only simple alcohols, polyols and
cyclic and aromatic alcohols, but also many simple carbohydrates using pyrophosphate as phosphate donor with a predominant regioselectivitity in favor of primhydroxy groups, only PhoN-Se converts sec-alcohols at low rates [607]. On the
contrary, stereoselectivities observed so far were modest.
Regioselective monophosphorylation of diols using PhoN-Sf, PhoN-Se and
PiACP (from Prevotella intermedia) using PP i , PPP i or polyP as phosphate donor
revealed that on short-chain diols, only mono-phosphorylation occurred,
bis-phosphates were formed to a small extent with long-chain analogs. Exclusive
O-phosphorylation was observed with an aminoalcohol [608].
Nucleotides are not only important intermediates for the synthesis of pharmaceuticals, but they also are widely used as flavor-enhancers and are thus produced
on industrial scale (~16,000 t/a). Among them are inosine- (5-IMP) and guanosine
5
0 -monophosphate (5-GMP), whose biological activity depends on the position of
the phosphate group: Whereas the 2
0 - and 3
0 -monophosphates are tasteless, the
5
0 -regioisomer is responsible for the ‘umami’-taste.
21 As nucleosides, such as
inosine and guanosine can be efficiently produced by fermentation, access to the
corresponding 5
0 -nucleotides depends on regioselective phosphorylation.
Non-specific acid phosphatases from Morganella morganii [609] and Escherichia
blattae [610] were employed in trans-phosphorylation using PP i as cheap phosphate
donor. In order to meet the requirements for an industrial process, the native
enzymes were mutated for enhanced binding of inosine by the phospho-enzyme
intermediate, which favors trans-phosphorylation, while undesired hydrolysis
is largely suppressed. The S72F/G74D/I153T-triple mutant was able to produce
140 g L
À1 of inosine 5
0 -phosphate with a yield of 71% from inosine (Scheme 2.82).
Table 2.2. Regioselective phosphorylation of diols and aminoalcohols using phosphatases
Substrate
Conc. [mM]
Enzyme
Product
Conc. [mM]
HO
OH
500
PhoN-Sf
mono/bis-P >99/<1
176
OH
HO
300
PhoN-Se
mono/bis-P 90/10
120
HO
OH
500
PhoN-Sf
mono/bis-P 90/10
305
HO
OH
500
PhoN-Sf
mono/bis-P 85/15
187
H 2 N
OH
500
PhoN-Se
O-P versus N-P >99/<1
85
21 Together with sweetness, saltiness, bitterness and sourness, ‘umami’ or ‘savory’-taste is one of
the five basic tastes.
114
2 Biocatalytic Applications
enterica (PhoN-Se) [606] phosphorylate not only simple alcohols, polyols and
cyclic and aromatic alcohols, but also many simple carbohydrates using pyrophosphate as phosphate donor with a predominant regioselectivitity in favor of primhydroxy groups, only PhoN-Se converts sec-alcohols at low rates [607]. On the
contrary, stereoselectivities observed so far were modest.
Regioselective monophosphorylation of diols using PhoN-Sf, PhoN-Se and
PiACP (from Prevotella intermedia) using PP i , PPP i or polyP as phosphate donor
revealed that on short-chain diols, only mono-phosphorylation occurred,
bis-phosphates were formed to a small extent with long-chain analogs. Exclusive
O-phosphorylation was observed with an aminoalcohol [608].
Nucleotides are not only important intermediates for the synthesis of pharmaceuticals, but they also are widely used as flavor-enhancers and are thus produced
on industrial scale (~16,000 t/a). Among them are inosine- (5-IMP) and guanosine
5
0 -monophosphate (5-GMP), whose biological activity depends on the position of
the phosphate group: Whereas the 2
0 - and 3
0 -monophosphates are tasteless, the
5
0 -regioisomer is responsible for the ‘umami’-taste.
21 As nucleosides, such as
inosine and guanosine can be efficiently produced by fermentation, access to the
corresponding 5
0 -nucleotides depends on regioselective phosphorylation.
Non-specific acid phosphatases from Morganella morganii [609] and Escherichia
blattae [610] were employed in trans-phosphorylation using PP i as cheap phosphate
donor. In order to meet the requirements for an industrial process, the native
enzymes were mutated for enhanced binding of inosine by the phospho-enzyme
intermediate, which favors trans-phosphorylation, while undesired hydrolysis
is largely suppressed. The S72F/G74D/I153T-triple mutant was able to produce
140 g L
À1 of inosine 5
0 -phosphate with a yield of 71% from inosine (Scheme 2.82).
Table 2.2. Regioselective phosphorylation of diols and aminoalcohols using phosphatases
Substrate
Conc. [mM]
Enzyme
Product
Conc. [mM]
HO
OH
500
PhoN-Sf
mono/bis-P >99/<1
176
OH
HO
300
PhoN-Se
mono/bis-P 90/10
120
HO
OH
500
PhoN-Sf
mono/bis-P 90/10
305
HO
OH
500
PhoN-Sf
mono/bis-P 85/15
187
H 2 N
OH
500
PhoN-Se
O-P versus N-P >99/<1
85
21 Together with sweetness, saltiness, bitterness and sourness, ‘umami’ or ‘savory’-taste is one of
the five basic tastes.
114
2 Biocatalytic Applications