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Phytase with wide ranging biochemical properties has long been utilized in a multitude of industries, even so, thermostability plays a crucial factor in choosing the right phytase in a few of the sectors. Mesophilic phytases are not considered to be a viable option in the feed industry owing to its limited stability in the required feed processing temperature. In the recent past, inclusion of thermostable phytase in fuel ethanol production from starch based raw material has been demonstrated with economic benefits. Therefore, considerable emphasis has been placed on using complementary approaches such as mining of extremophilic microbial wealth, encapsulation and using enzyme engineering for obtaining stable phytase variants. This article means to give an insight on role of thermostable phytases in feed and fuel industries and methods for its development, highlighting molecular determinants of thermostability.

L-Asparaginase is an antineoplastic agent that selectively decreases the level of L-asparagine in blood and diminishes the proliferation of the cancerous cells. L-Asparaginases from Escherichia coli are widely used for clinical application because of their high substrate specificity and limited glutaminase activity. L-Asparaginase II-encoding gene ansB was isolated by excluding the native signal from E. coli MTCC 739, cloned in frame with pelB leader sequence of prokaryotic expression vector pET20b and expressed in E. coli DE3 cells. Overexpression of recombinant protein was achieved with an optimized final concentration of 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was expressed as soluble protein. The recombinant protein contained hexahistidine tag at C-terminus and was purified using nickel-nitrilotriacetic acid chromatography. Enzymatic properties such as optimum temperature, pH and the effect of temperature on the stability of L-asparaginase II from E. coli MTCC 739 were determined and the purified protein showed an optimum activity at 37 °C and pH=6., L-asparaginaza je antineoplastični agens koji selektivno smanjuje razinu L-asparaginaze u krvi i sprečava proliferaciju kanceroznih stanica. L-asparaginaze iz bakterije Escherichia coli koriste se za klinička ispitivanja zbog njihove specifičnosti za supstrat i ograničene aktivnosti glutaminaze. Gen ansB za kodiranje L-asparaginaze II izoliran je iz E. coli MTCC 739, kloniran pomoću pelB sekvencije prokariotskog vektora pET20b i izražen u stanicama E. coli DE3. Pojačana je ekspresija rekombinantnog proteina postignuta pomoću 10 μM izopropil β-D-1-tiogalaktopiranozidaze, pri čemu je izraženi protein bio topljiv, a na C završetku imao je heksahistidinsku oznaku. Rekombinirani je protein pročišćen kromatografijom pomoću nikal(II)-nitrilotrioctene kiseline. Ispitani su ovi parametri: optimalna temperatura i pH-vrijednost za aktivnost enzima, te utjecaj temperature na njegovu stabilnost. Utvrđeno je da je pročišćeni protein imao optimalnu aktivnost pri 37 °C i pH=6.

Microbial phytases, especially from fungal and bacterial sources, have received much attention as food additives in human nutrition and as feed supplements for monogastric animals. An effective expression screening method for recombinant production of this enzyme on a small scale is industrially desirable. An effort has been made in this work to clone and express phytase genes from Aspergillus sp. and Escherichia coli with the selected host, vector and inducer combination. Albeit the formation of insoluble inclusion bodies by fungal phytase, recombinant E. coli appA was effectively expressed in a cost-effective manner in the periplasm of BL21plysS using an inducer concentration of 0.01 mM in 4 h of growth. Enzyme was purified in three consecutive steps and functional studies were carried out., Mikrobne fitaze, osobito iz plijesni i bakterija, imaju vrlo važnu ulogu u prehrambenoj industriji kao aditivi i dodaci prehrani monogastričnih životinja. Za industrijsku je proizvodnju poželjno pronaći učinkoviti postupak ekspresije gena za dobivanje rekombinantnih fitaza. U radu je prikazan postupak kloniranja i ekspresije gena za proizvodnju fitaza iz plijesni vrste Aspergillus i bakterije Escherichia coli, s pomoću pažljivo odabrane kombinacije domaćina, vektora i induktora. Iako su pritom nastali netopljivi agregati (tzv. inkluzije), rekombinantni gen E. coli appA uspješno je i bez većih troškova izražen u periplazmi stanica BL21plysS, uz koncentraciju induktora od 0,01 mM, tijekom 4 sata rasta. Enzim je pročišćen u tri uzastopna koraka, te su ispitane njegove značajke.