(88b) Kinetic Model of Enzymatic Hydrolysis in Liquid – Liquid System for Continuous Processes

Lipases enzyme (EC 3.1.1.3; triacylglycerol hydrolases) is one of
the promising enzymes for industrial application. It is known for the splitting
of triglycerides, or a more popular application is the enzymatic production of
biodiesel. The substrate is the triglycerides in different vegetable oils –
sunflower oil, rapeseed oil or even cooking oil, which hydrolyzed into free
fatty acids (FFA) or transesterified into fatty acid alkyl esters
(FAAE) as biodiesel.

Compared to conventional processes, enzymatic hydrolysis holds the
advantage of mild process conditions, which in turn not only saves energy but
also ensures the stability of temperature-sensitive products, for example, the
production of non-trans fatty acids. Despite the advantages, industrial
application of such enzymatic system is so far not feasible, due to the high
cost of biocatalysts. An optimum process condition is necessary to have a high
turnover number of the enzyme, to ensure the highest yield possible per amount
of enzyme.

A continuous process is being developed for the enzymatic hydrolysis
of triglycerides. The unique property of this enzyme is its interfacial
activity at the water-oil interface. The higher the surface area for these
enzymes to adsorb, the faster the reaction rate.

A stirred tank reactor is used to create water in oil emulsion, to
increase the reaction rate. The phases are then separated in a gravity settler
to remove the primary product from the oil phase, and to recycle the enzymes in
the aqueous phase while separating the Glycerol as a secondary product.

To develop this continuous process, a kinetic model
is needed to predict the residence time. The model of the interfacial reaction
of lipases has to include two aspects: 1. The influence of two substrates and
two products to the reaction equilibrium and 2. The dependence of the reaction
rate to the availability of surface area in the emulsion. Ping-Pong Bi-Bi
mechanism is modified with adsorption kinetic to model the limitation of surface
area and the adsorption kinetics of lipases on the interface.

The first step of the reaction is adsorption of enzyme on interface
area, creating an activated enzyme (E*). 
This activated enzyme can react with the first substrate, TG/DG/MG –
depending on the enzyme's substrate specificity, into an enzyme complex (EAc), which will be hydrolyzed into Fatty Acid (P). By
splitting the reaction into these 5 different steps, it is possible not only to
predict the initial reaction rate but also to model the accumulation of
side/intermediate products and their inhibition to the reaction. This
prediction is needed to conduct the reaction in the continuous process, as the
continuous kinetics is different from batch kinetics, due to continuous removal
of glycerol from the aqueous phase.

The model parameters are fitted with experimental data in batch
reactors, and then validated for different scales: 0.4L, 1L and 13L. The model
is able to predict the reaction not only based on enzyme concentration and
surface area, but also the limitation of Triglycerides, water, and glycerol.

By using this
model, it is possible to predict the reaction rate for different water-oil
ratio, enzyme concentration, and stirring rate. In the continuous process,
further optimization is needed between the stirring rate, temperature, water to
oil ratio and residence time:

·        
Higher stirring rate results in
a higher reaction rate, but also longer water oil separation.

·        
Higher temperature increases
the reaction rate, reduces water – oil separation time by decreasing the
viscosity of the continuous phase, but at the same time induces faster enzyme
deactivation.

·        
Higher water to oil ratio
increases the reaction rate, promotes water – oil separation.

The
abovementioned parameters are being balanced in the 13L continuous process, to
determine the optimum condition for long term processes. Successful application
of biocatalysts in such continuous system can demonstrate the feasibility of
the enzymatic process as a greener alternative to the conventional processes.
With a proper enzyme retention system, it is possible to develop a continuous
process with long term reusability of the enzymes, which in turn reduce the
cost of catalysts in the system.