161
(DiCosimo et al. 2013). However, as a main interference, immobilized lipases cannot undergo interfacial activation via interaction with oil or solvent interfaces
because oil drops are unable to penetrate inside the porous structure of the catalyst.
The lack of interfacial activation can be compensated by promoting the activation of
lipase during its immobilization. An innovative methodology was reported by Filice
and Marciello (2013), expecting the hyperactivation of Rhizomucor miehei lipase
(RML) through different concentrations of different detergents and the subsequent
immobilization of the most active open form via intense multipoint anion exchange.
RML immobilized inside porous supports by means of this strategy becomes highly
stable, very active, and selective for the mild hydrolysis of fish oils with EPA production without interaction with interfaces and of aggregation phenomena. In this
way, interfacial activation of soluble lipases on oil drops is not really necessary
because a similar activation can be achieved via a careful non-natural hyperactivation of the immobilized enzyme.
Another strategy to enhance the production level of an enzyme is by presenting
more copies of the relevant gene into the concerned organism. For instance, the penicillin-G-oxidase-encoding gene from E. coli was integrated into the vector pBR322
and then the recombinant plasmid was introduced into E. coli. The recombinant
strain produced considerably higher quantities of the enzyme than their original/parent strain, thereby reducing the production costs and also enhancing the purity of the
enzyme. The applications of recombinant DNA technology are considered to be
much more advantageous as well as diverse than that for modifying amino acid
sequences of enzymes. Such enhanced yields are economically significant due to
enhanced volumetric productivity and reduced downstream processing costs, and
also due to the fact that the crude enzymes are more pure. Robas et al. (1993) used
the same principle to enhance the activity of penicillin-G-amidase in E. coli.
Many of the commercially available enzymes are produced by genetically modified microorganisms (GMMs) (Pedersen et al. 1995), which provide enzymes with
higher specificity and purity. Because of an efficient separation process, the GMMs
are removed completely from the final enzyme product. Novozymes uses the strategy of GMMs for marketing a wide range of enzymes for various industrial purposes. Several enzyme preparations made by GMMs are currently used in the
beverage industry. NovoShape™, containing a pure pectin esterase, helps retain the
original shape and structure of individual fruit pieces during processing and thereby
offers a finished product that is more appealing (http://www.novozymes.com).
Pectinex
®
SMASH, containing a variety of different pectinases, is used for treating
apple and pear mash for higher yield and capacity (http://www.novozymes.com).
6.5
Challenges in Enzyme Engineering
Enzyme engineering has developed as an essential mechanism to overcome the constraints of using native enzymes as biocatalysts. Latest advances in enzyme engineering have mainly aimed at applying directed evolution to enzymes, especially
important for organic synthesis, such as monooxygenases, ketoreductases, lipases,
or aldolases in order to improve their activity, enantioselectivity, and stability.
6 Enzyme Engineering
(DiCosimo et al. 2013). However, as a main interference, immobilized lipases cannot undergo interfacial activation via interaction with oil or solvent interfaces
because oil drops are unable to penetrate inside the porous structure of the catalyst.
The lack of interfacial activation can be compensated by promoting the activation of
lipase during its immobilization. An innovative methodology was reported by Filice
and Marciello (2013), expecting the hyperactivation of Rhizomucor miehei lipase
(RML) through different concentrations of different detergents and the subsequent
immobilization of the most active open form via intense multipoint anion exchange.
RML immobilized inside porous supports by means of this strategy becomes highly
stable, very active, and selective for the mild hydrolysis of fish oils with EPA production without interaction with interfaces and of aggregation phenomena. In this
way, interfacial activation of soluble lipases on oil drops is not really necessary
because a similar activation can be achieved via a careful non-natural hyperactivation of the immobilized enzyme.
Another strategy to enhance the production level of an enzyme is by presenting
more copies of the relevant gene into the concerned organism. For instance, the penicillin-G-oxidase-encoding gene from E. coli was integrated into the vector pBR322
and then the recombinant plasmid was introduced into E. coli. The recombinant
strain produced considerably higher quantities of the enzyme than their original/parent strain, thereby reducing the production costs and also enhancing the purity of the
enzyme. The applications of recombinant DNA technology are considered to be
much more advantageous as well as diverse than that for modifying amino acid
sequences of enzymes. Such enhanced yields are economically significant due to
enhanced volumetric productivity and reduced downstream processing costs, and
also due to the fact that the crude enzymes are more pure. Robas et al. (1993) used
the same principle to enhance the activity of penicillin-G-amidase in E. coli.
Many of the commercially available enzymes are produced by genetically modified microorganisms (GMMs) (Pedersen et al. 1995), which provide enzymes with
higher specificity and purity. Because of an efficient separation process, the GMMs
are removed completely from the final enzyme product. Novozymes uses the strategy of GMMs for marketing a wide range of enzymes for various industrial purposes. Several enzyme preparations made by GMMs are currently used in the
beverage industry. NovoShape™, containing a pure pectin esterase, helps retain the
original shape and structure of individual fruit pieces during processing and thereby
offers a finished product that is more appealing (http://www.novozymes.com).
Pectinex
®
SMASH, containing a variety of different pectinases, is used for treating
apple and pear mash for higher yield and capacity (http://www.novozymes.com).
6.5
Challenges in Enzyme Engineering
Enzyme engineering has developed as an essential mechanism to overcome the constraints of using native enzymes as biocatalysts. Latest advances in enzyme engineering have mainly aimed at applying directed evolution to enzymes, especially
important for organic synthesis, such as monooxygenases, ketoreductases, lipases,
or aldolases in order to improve their activity, enantioselectivity, and stability.
6 Enzyme Engineering