ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 2, с. 135-138
UDK 577.15+579.222
REGIOSELECTIVE HYDROLYSIS OF ACETATES IN THE PRESENCE
OF DIFFERENT YEAST STRAINS
© 2014 J. Krzyczkowska, E. Majewska, E. Bia+ecka-Florjanczyk
Department of Chemistry, Faculty of Food Sciences, Warsaw University of Life Sciences - SGGW, 16602-787 Warsaw, Poland e-mail: jolanta_krzyczkowska@sggw.pl Received August 20, 2013
The model compound, hexane-1,2-diol diacetate, was hydrolyzed in the presence of supernatant obtained after cultivation of 4 yeast strains: Pichia jadinii, Rhodotorula glutinis and Yarrowia lipolytica KKP 379 and Saccharomyces cerevisiae 102 to evaluate the type of catalysis. The regioselectivity of extracellular enzymes as a function of hydrolysis towards primary and secondary acetic acid ester groups was monitored. The enzymes secreted by P. jadinii, R. glutinis and Y. lipolytica KKP 379 exhibited high regioselectivity towards primary position, while those from S. cerevisiae showed practically no discrimination between the ester groups.
DOI: 10.7868/S0555109914020111
Many biocatalysts are used in protection/depro-tection chemistry which is indispensable in the construction of complex polyfunctional molecules [1]. Among them, the hydrolytic enzymes are particularly useful for regioselective acylation/deacylation of poly-hydroxyl compounds, which can be achieved in 2 ways: involving the selective monoacylation of the polyhydroxyl compounds, and catalyzing the selective removal of a single acyl group from the polyacelated compounds. Hydrolytic enzymes, especially lipases, have been widely applied in regioselective transformations in organic synthesis due to their specifity and ability of accommodation toward a wide range of substrates. Although the natural substrates of lipases are triacylglyceroles, which have very low solubility in water, these enzymes can catalyze the hydrolysis and synthesis of other ester bonds as well [2].
The regioselective capabilities of lipases have been applied in solving problems of different alcoholic group recognition within the same molecule. In this case the discrimination between the primary and secondary hydroxyls is possible not only in triacylglycer-ols [3] but also in carbohydrates [4, 5] as well as in glycosides [6] or flavones [7]. Furthermore, lipases are able to interact with substrates that are structurally unrelated to their natural targets — for example, the lipase catalysed deacetylation of some polyphenols [8], phenolic antioxidants [9] and transesterification of naphtyl alcohols [10]. Yeast is an excellent source of various enzymes (especially hydrolases and oxi-doreductases) applied in biocatalytic processes. These enzymes can be employed in a purified form or without isolation — the yeast whole cells are often used as catalysts in many reactions [11, 12].
The regioselectivity issue in hydrolysis of ester bonds is interesting in connection with diols modifica-
tion. Monoesters of diols find many applications in pharmaceutical, cosmetic and food industries. So far, in hydrolysis and transesterifications of esters derived from diols only isolated and commercially available lipases were used [13] and less attention has been focused on the evaluation of the potential lipases from different yeasts.
In our previous studies on the participation of catalytic activity of yeasts in the hydrolysis of alkane car-boxylic acids, phenyl esters were examined [12] and the impact of medium composition for this reaction was determined [14]. Some of yeast species were used as a catalyst in the synthesis of 2-phenylethyl acetate [15], an essential aroma component.
The aim of this study was to evaluate the selectivity of different yeast strains (of genera Yarrowia, Rhodotorula, Pichia and Saccharomyces) towards primary and secondary acetyl groups.
MATERIALS AND METHODS
Materials. Hexane-1,2-diol and acetic anhydride were obtained from Sigma-Aldrich (USA); chloroform and 96% ethyl alcohol were purchased from Polish Chemicals Reagents S.A. (Poland). All these reagents were analytical grade and used without further purification. Yeast extract, peptone, glucose and agar were supplied by Department of Enzymes and Peptones BTL (Poland). Pichia jadinii, Rhodotorula glutinis, Saccharomyces cerevisiae 102 were purchased from the microbial culture collection of the Department of Food Biotechnology and Microbiology of Warsaw University of Life Sciences (Poland). Yarrowia lipolytica KKP 379 was obtained from the microbial culture collection of the Agricultural and Food Biotechnology Institute in Warsaw (Poland). The yeast
CH3COO-CH2 HO- CH2 CH3COO" CH2
l HO l l
CH3COO-CH -^ar CH3COO~CH + HO"CH
(CH2)3 (CH2)3 (CH2)3
CH3 CH3 CH3
Hexane-1,2-diol diacetate 2-acetoxyhexan-1-ol 1-acetoxyhexan-2-ol
Isomer A Isomer B
Fig. 1. Scheme of hydrolysis of hexane-1,2-diol diacetate.
strains were maintained on yeast extract-peptone-glu-cose-agar slants (YPG medium with agar, added in an amount of 20 g/L), which were stored at 5°C between transfers. The composition of YPG medium is given below.
Synthesis of hexane-1,2-diol diacetate. Hexane-1,2-diol diacetate was synthetized from hexane-1,2-diol (6.0 g) and acetic anhydride (15.5 g) [16]. The product was extracted from the reaction mixture with chloroform (3 x 50 mL) and distilled (boiling point 85°C; 2 mm Hg). The purity of product after the distillation was analyzed by gas chromatography.
Supernatant production. Precultures of yeasts were prepared in two steps. First, cells from the agar slants were transferred into a 100 mL Erlenmeyer flask containing 50 mL YPG medium (composition of medium g/L: yeast extract — 10; peptone — 20 and glucose — 20; pH 5.0) and incubated in a shaker incubator at 28°C and 200 rpm for 48 h. Then, 15 mL of this medium was added to a 250 mL Erlenmeyer flask containing 85 mL YPG cultivation medium, and grown at the same conditions for 24 h. 1 mL of this preculture was used to inoculate a 250 mL Erlenmeyer flask containing a working volume of 100 mL YPG medium at 28°C and 200 rpm agitator speed. After 48 h of cultivation, cells were separated by centrifuga-tion (6000 x g for 10 min at 4°C) and supernatant was transferred to the hydrolysis medium.
Hydrolysis. Hydrolysis of hexane-1,2-diol diacetate (1.5 mmol dissolved in 0.5 mL of ethyl alcohol) was carried out in Erlenmeyer flask (500 mL) with 60 mL of proper supernatant. The reaction was allowed to proceed at room temperature with shaking at 200 rpm for 5 h. Samples of reaction mixtures were extracted with chloroform and analyzed by gas chroma-tography.
Chromatographic analysis. Substrate and product contents were monitored by gas chromatography (GC — Shimadzu, Japan) using a capillary column (BPX-70; 30 m x 0.22 mm; SGE Analytical Science, United Kingdom) and a flame ionisation detector (250°C; Shimadzu, Japan). A flow of 1.10 mL/min carrier gas (N2) was used. After injection of samples, the temperature of the column oven was kept constant for 1 min (60°C) and then linearly increased as 10°C/min to 200°C and kept at 200°C for 5 min.
Breakdown products of ester were identified by mass spectrometry (MS) (GCMS 2010; Shimadzu, Japan). The temperature of the column (BPX 70) oven was kept constant for 1 min (55°C), and then linearly increased as 4°C/min to 220°C and kept at 220°C for 3 min.
MS data (m/z) were:
hexane-1,2-diol diacetate: 202 (molecular ion), 129, 117, 100, 86, 82;
isomer I: 159 (molecular ion), 129, 117, 87, 74, 69;
isomer II: 159 (molecular ion), 103, 87, 73, 69.
RESULTS AND DISCUSSION
Yeast excrete several hydrolytic enzymes (lipases and esterases EC 3.1.1) and the most of them are expressed extracellularly [17—19]. Hexane-1,2-diol was chosen as a model compound due to its amphiphilic character and relatively low water solubility, which enabled lipases to participate in the reaction, taking advantage of all hydrolytic enzymes produced. The hydrolysis reactions were carried out in the presence of cell-free supernatant received after cultivation of yeast, without isolation of enzymes.
The hydrolysis reaction of hexane-1,2-diol diacetate may lead to 2 different monoacetates (Fig. 1), depending on which ester bond will break down, as well as to hexane-1,2-diol in case of the hydrolysis of both ester bonds. Isomer A is formed when the primary ester bond is hydrolyzed, isomer B is the product of the secondary ester bond cleavage.
As can be seen at the Fig. 2a, 2b and 2c the hydrolysis reaction of hexane-1,2-diol diacetate in the presence of cell-free supernatant obtained after cultivation of such yeast strains as P. jadinii, R. glutinis and Y. li-polytica KKP 379, had a highly regioselective course. Primary ester bond was hydrolyzed predominantly. Hexane-1,2-diol diacetate has almost disappeared after 5 h of incubation, but the product of the hydrolysis of both ester bonds (hexane-1,2-diol) had not been detected.
Since the hydrolysis was carried out for acetic acid esters, one can suppose that enzymes taking part in this reaction are most likely esterases which are active towards esters of short-chain carboxylic acids. On the other hand, lipases can also catalyze this reaction and their ability to hydrolyse acetyl groups is well docu-
nPHKHA^HAH EHOXHMHH H MHKPOEHO^Oratf tom 50 № 2 2014
REGIOSELECTIVE HYDROLYSIS OF ACETATES IN THE PRESENCE
137
%
100 80 60 40 20
0
%
100 80 60 40 20
(а)
2 3
(c)
........*2
%
100
a2 80
60
40
-3 20 1
5 0
h %
100 80 60 40
3 20 1
5 0 h
(b)
-—a2
2 3
(d)
5 h
5 h
Fig. 2. Composition of reacting mixture (%) during hydrolysis of hexane-1,2-diol diacetate in the presence of enzymes produced by: P. jadinii (a), R. glutinis (b), Y. lipolytica KKP 379 (c), S. cerevisiae 102 (d). 1 — diester; 2 — isomer A; 3 — isomer B.
1
1
0
1
1
mented [7—8]. In literature no reports on regioselec-tivity of yeast extracellular esterases were found.
The ratio between isomers A and B was about 6 : 1 for a
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.