Development and scale-down of enzyme analysis methods for high throughput screening of important soil enzymes

Enzymes are generally accepted as the "Swiss-army knife" of sustainable bio-economy and as such, they represent an economical significance. Chief among those amylase, protease, phosphatase and urease for which various studies about their production methods is an attractive topic in biotechnology research. The heart of industrial biotechnology is the availability of the producer (micro)organism. While the recombinant-route involving the use of advanced molecular techniques e.g. synthetic biology toolbox are of great interest, the immense undiscovered potential of nature and natural producers is very appealing, not only for economical purpose, but also from scientific point of view, allowing to discover possibly new mechanism even for already known processes. To materialize this potential, fast and affordable, high throughput screening methods that will allow identification of producers of desired enzymes among large number of environmental samples, need to be developed. This requires in turn, detailed study of enzyme properties, modification of available methods or development of novel methods if necessary. This thesis aims to set up such high throughput methods for four aforementioned enzymes. The developed, scaled-down methods are based on a combination of spectroscopic and colorimetric approaches, with automatization and miniaturization, on 96-micro titer well plates (MTP). The methods are first tested using type strains for which the activity are known. Later, the developed methods are used to screen the in house available collection, consisting of 537 unknown microorganisms. Using this method new strong enzyme producers were discovered and reported.87 isolates was observed as urease positive and 76 isolates was observed as amylase positive. Among those organisms 6ys29 is both urease and amylase positive. 15 isolates was detected as protease producers. 5ys19, 7ys60, 7ys66 are both protease and urease, 6ys29 is amylase and urease positive microorganism. 7ys48 is protease and amylase positive. 6ys59, 6ys33, 5ys5 are phosphatase and amylase, 7ys39, 5ys23, 7ys76, 5ys42 are urease and phosphatase positive isolates.