BIOCONVERSION OF GLYCEROL INTO GLYCERIC ACID
CATALYZED BY PQQ-DEPENDENT ALCOHOL DEHYDROGENASE
Ana Chaleckaja1, Lidija Tetianec1,2, Juozas Kulys1,2, Liucija Marcinkeviciene1, Jonita
Stankeviciute1, Rolandas Meskys1*
1
Institute of Biochemistry, Life Science Center, Vilnius University, Sauletekio al. 7, LT-10257, Vilnius, Lithuania
Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Faculty of Fundamental
Sciences, Sauletekio al. 11, LT-10223, Vilnius, Lithuania
[email protected]
2
Glycerol is a major byproduct in the biodiesel manufacturing process. Biodiesel production generates about 10% of
glycerol [1]. Very pure glycerol is an important stock material for the food products, pharmaceutical, tobacco or cosmetics
industries, but obtained as biodiesel synthesis trash is shoddy, with impurities and its purification is expensive. Thus, the
expansion of the biodiesel industry is confront with the problem of the lack of an economically favorable way in which
glycerol is processed into higher value-added products [2,3]. Therefore the biodiesel producers must seek alternative
methods for its conversion. One of the promising applications of glycerol is its bioconversion to glyceric acid, which is
important compound as raw materials for chemical products, such as bioplastics, pharmaceuticals for acceleration of
alcohol metabolism or liver disease treatment and cosmetics [4].
The task of our investigation is to create the prototype of a bioreactor for the oxidation of glycerol into glyceric acid.
Electroenzymatic method was employed to achieve the goal, and the PQQ-dependent alcohol dehydrogenase (ADH IIG)
was the key enzyme in conversion of the substrate along with the electrochemically oxidized mediator.
The reactivity of the enzyme with a compound defines the efficiency of the compound in the conversion scheme.
The dependences of initial reaction rate on electron acceptor’s (ferricyanide, N,N'-dimethyl-4,4'-azopyridinium methyl
sulfate (MAZP) and -1-(NN'-dimethylamine)-4-(4-morpholine)benzene (AMB)) concentrations were analyzed and the
bimolecular reactivity constant values were calculated by applying the ping-pong enzyme action scheme. For different
electron acceptors the bimolecular ADH IIG and electron acceptor reactivity constant (k ox) values varied from
(4.7±0.1)·104 M−1s−1 to (2.8±1.0)·105 M−1s−1.
The MAZP was used as electron acceptor for reduced ADH IIG in order to assess the degree of conversion (R) of
substrates - glycerol and glyceraldehyde. The decrease of the concentration of MAZP oxidized form in the presence of
ADH IIG and substrates (glycerol, D,L-glyceraldehyde, D-glyceraldehyde or L- glyceraldehyde) was monitored
spectrophotometrically. When glycerol was used as a substrate the obtained R-value was 2. This indicates that in the
reaction mixture all glycerol was oxidized into glyceric acid. For racemic glyceraldehyde or its optically pure enantiomers
the R equals to ~1, indicating that the oxidation of these substrates occurs via one stage to corresponding glyceric acid.
Ferricyanide, MAZP and AMB were used as an electrochemical oxidized mediators for ADH IIG catalyzed glycerol
conversion. The formation of glyceric acid and glyceraldehyde, which are the products of glycerol oxidation, were
revealed by monitoring their 2-nitrophenylhidrazine derivatives by HPLC. The larger glycerol conversion yields were
obtained using the larger amounts of mediator MAZP and KOH to neutralize the glyceric acid. During the optimization
of prototype of reactor the TTN’s of up to 37 for MAZP and 2264 for enzyme were achieved.
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(2012).
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Chilean journal of agricultural reseach 71(3), 469-475 (2011).
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International Meeting Sponsored by ASABE Oregon Convention Center Portland, Oregon 9 - 12 July, 1-16 (2006).
[4] H. Habe, T. Fukuoka, D. Kitamoto, and K. Sakaki, Biotransformation of Glycerol to D-Glycericacid by Acetobacter tropicalis, Applied Microbiology
and Biotechnology, 81, 1033–1039 (2009).