1. INTRODUCTION
1.7 Esterification and transesterification reactions catalyzed by cutinase
Short chain fatty acids esters are commonly used in the manufacturing of flavors and fragrances because of their fruity odor. These esters are in high demand and are widely used in the food, beverage, cosmetic, and pharmaceutical industries. Fatty acid esters of alcohols are also of increasing economic interest in many industries involving a wide range of applications; viz., bio-carburants, biosurfactants, biolubricants, solvents, hydraulic and drilling fluids, dispersing agents, cosmetics (Dossat et al., 2002) and biodiesel (Xie and Ma, 2010).
Although flavor esters are currently produced by chemical synthesis, there is an increasing inclination towards natural flavors, rather than chemically synthesized flavors. However, the same flavor and fragrance esters prepared by enzymatic synthesis may be labeled as “natural”
(Gillies et al., 1987; Larios et al., 2004). Lipases and esterases are commonly used for the enzymatic production of a wide range of ester products in non-conventional media.
Nevertheless, most of the lipases have higher affinity towards longer–chain length substrates, and low molecular weight substrate may have some inhibitory effect on enzymes (Abbas and Comeau, 2003; Hari-krishna and Karanth, 2002). In case of esterases, except few, most esterases loss their reactivity when the acyl chain length of substrate is more than two (Macarie and Baratti, 2000; Torres et al., 2009). Recently, cutinases (Cunnah et al., 1996; De Barros et al., 2009a; De Barros et al., 2010a; Dutta and Dasu, 2011; Pinto-Sausa et al., 1994;
Sarazin et al., 1992; Sarazin et al., 1995; Sebastiao et al., 1993; Serralha et al., 2001) were employed for the synthesis of some alkyl esters. Cutinases are believed to be a group of hydrolytic enzyme intermediate between lipase and esterase, which are capable of hydrolyzing various soluble esters as well as emulsified triacylglycerol. As the esterification reaction is reversible, the enzymes can also be employed to catalyze the formation of alcohols and fatty acids from esters. However, in aqueous solutions, the hydrolysis reaction is favored over synthesis reaction as the equilibrium is strongly shifted towards the starting reagents and esters cannot be synthesized. To overcome this difficulty, reaction media containing very small amounts of water or comprised of organic solvents can be used to bring about a
INTRODUCTION
8
chemical equilibrium shift towards ester. Recently, few studies on the production of methyl/ethyl ester of oil mixtures have also been carried out using cutinase from F. solani pisi (Badenes et al., 2010a; Badenes et al., 2011).
1.8 Cutinase catalytic site
To date, five cutinase crystal structures had been resolved, this includes F. solani, A. oryzae, G. cingulate, Cryptococcus and recently, T. alba (Kitadokoro et al., 2012; Kodama et al., 2009; Liu et al., 2009; Longhi et al., 1997; Martinez et al., 1992; Nyon et al., 2009).
The overall structural studies of cutinase reviles that all the cutinases belong to the class of serine esterases and to the super family of the α/β hydrolases in which the nucleophilic serine is located at the center of an extremely sharp turn between a central β-strand surrounded by α-helices. The consensus sequence G-X-S-X-G with active serine in the catalytic triad is seen in all the cutinases. They have solvent exposed catalytic triad “Ser-Asp-His” and an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups (Nicolas et al., 1996). One to two disulfide bridges are seen in cutinase that helps to the stabilization of the global molecular folding of cutinase. The binding of cutinase to interfaces seems not to require a main-chain rearrangement, as in the case of lipases, but only the reorientation of few lipophilic side chains.
1.9 Cutinase mechanism of action
Cutinases show catalytic machinery similar to those present in serine proteases. The catalytic triad is a coordinated structure consisting of three essential amino acids: histidine, serine and aspartic acid, which play an essential role in the cleaving ability of the cutinase.
The general catalytic mechanism is shown in the Fig. -. In brief, the reaction occurs as follows. The substrate binds to the surface of the cutinase such that the scissile bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond positioned near the nucleophilic serine. The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the ester bond of the substrate. A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group, thus coordinating the attack of the ester bond. The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the nitrogen atom mentioned above much more electronegative. The reaction stabilization is also brought by additional amino acids, Met and
TH-1214_KHEGDE
INTRODUCTION
Tyr (Bacterial cutinase) or Gly and Ser (Fungal cutinase) in the vicinity of the active site, by formation of oxyanion hole.
1.10 Enzyme reaction kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction is investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of particular enzyme, its role in catalysis, how its activity is controlled, and how an inhibitor might inhibit the enzyme.
Unlike uncatalysed chemical reactions, enzyme-catalysed reactions display saturation kinetics. For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration; the enzyme molecules are largely free to catalyse the reaction, and increasing substrate concentration means an increasing rate of enzyme catalysis. However, at relatively high substrate concentrations, the reaction rate asymptotically approaches the theoretical maximum due to the fact that all the active sites of enzyme are almost occupied and the reaction rate is determined by the intrinsic turnover rate of the enzyme. The substrate concentration midway between these two limiting cases is denoted by KM.
Michaelis–Menten kinetics is one of best-known models of enzyme kinetics. The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate (v) to the concentration of a substrate (s). Its formula is given by,
V= (Vmax X [s])/(KM + [s])
Where, Vmax represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations, s is the substrate, The Michaelis constant Km is the substrate concentration at which the reaction rate is half of Vmax. Biochemical reactions involving a single substrate are often assumed to follow Michaelis-Menten kinetics.
However, multi-substrate reactions follow complex rate equations that describe how the substrates bind and in what sequence. Depending on the substrates involved and the bilding of substrates, it can be classified as, The Sequential Mechanism (Ordered Sequential Mechanism and Random Sequential Mechanism) and the Non-sequential or ‘‘Ping-Pong’’ Mechanism.
Inhibitors are compounds that decrease the rate of an enzyme-catalyzed reaction. The study of enzyme inhibition has enhanced our knowledge of specificity and the nature of functional
INTRODUCTION
10
groups at the active site. The activity of certain enzymes is regulated by a feedback mechanism such that an end product inhibits the enzyme’s function in an initial stage of a sequence of reactions. The action of an inhibitor on an enzyme can be described as either reversible or irreversible. In reversible inhibition, an equilibrium exists between the enzyme and the inhibitor. There are three important types of reversible inhibition: competitive inhibition, noncompetitive inhibition, and uncompetitive inhibition. In irreversible inhibitions, inhibition progressively increases with time. Complete inhibition results if the concentration of the irreversible inhibitor exceeds that of the enzyme.