Microbial enzymes are of great importance in the development of industrial

Microbial enzymes are of great importance in the development of industrial bioprocesses. such hurdles alternative bacterial host and expression systems are currently being examined including and or and have been produced and their potential has been reviewed [51 52 Microorganisms that can survive under extreme pH values could be good sources of thermoalkaliphilic enzymes like proteases and lipases particularly useful for applications as additives in laundry and dishwashing detergents [16 53 3 Strategies to Improve Properties of Microbial Enzymes The constantly expanding application of enzymes is usually creating a growing demand for biocatalysts that exhibit improved or new properties [46]. Although enzymes have favorable turnover numbers they do not necessary fulfill all process requirements and need further fine tuning to achieve industrial scale production. Among those hurdles are: substrate/product inhibition stability narrow substrate specificity or enantioselectivity [54]. Genetic modification is very important and recombinant DNA techniques have increased production by 100-fold [55]. Development of new/improved biocatalysts is usually a challenging and complex task (Physique 1). There are two major ways in which enzymes can be altered to adapt their functions to applied ends: (i) rational redesign of existing biocatalysts and (ii) combinatorial methods which search for the desired functionality in libraries generated at random. Figure 1 Discovery and development of biocatalysts. 3.1 Rational Design This approach includes site-directed mutagenesis to target Rabbit polyclonal to AHCYL1. amino acid substitutions thus requiring knowledge of detailed information about the 3-dimensional structure and chemical mechanism of the enzymatic reaction some of which may not be available. However the increasing growth of databases containing protein structures and sequences is usually helping to overcome Procoxacin this lack of information. Comparison of the sequence of a new biocatalyst identified Procoxacin in a screening program with the thousands deposited in the databases can identify related proteins whose functions or/and structures are already known. Because new enzymes have evolved in nature by relatively minor modification of active-site structures the goals of homology-driven experiments include engineering binding sites to fit different substrates as well as construction of new catalytic residues to modify functions and mechanisms [56]. A small number of variants are produced which are then screened. Although in many cases results are poor compared to natural enzymes there have been successes [57 58 Computational protein design starts with the coordinates of a protein main chain and uses a pressure field to identify sequences and geometries of amino acids that are optimal for stabilizing the backbone geometry [59]. Because of the amazing number of possible sequences generated the combination of predictive pressure fields and search algorithms is now being applied to functional Procoxacin protein design [60]. 3.2 Directed Evolution Combinatorial methods such as directed evolution create a large number of variants for screening for enantioselectivity catalytic efficiency catalytic rate solubility specificity and enzyme stability but do not require extensive knowledge about the enzyme. Directed evolution is a fast and inexpensive way of obtaining variants of existing enzymes that work better than naturally occurring enzymes under specific conditions Procoxacin [3 61 62 63 Directed evolution includes an entire range of molecular biological techniques that allow the achievement of genetic diversity mimicking mechanisms of evolution occurring in nature. It involves random mutagenesis of the protein-encoding gene by different techniques including the error-prone polymerase chain reaction (PCR) [64] repeated oligonucleotide directed mutagenesis [65] or chemical agents [66] among others. Error prone PCR accomplishes introduction of random point mutations in a populace of enzymes. Such molecular breeding techniques (DNA shuffling Molecular BreedingTM) allow random homologous recombination typically between parent genes with homology higher than 70% [67]. After cloning and expression a large collection of enzyme variants (104-106) is typically generated and is subjected to screening or selection. All Procoxacin the approaches mentioned above are not mutually unique as the fields of rational semi-rational and random redesign of enzymes are moving closer. Thus directed evolution techniques make use where possible of smaller.