Cold areas that comprise up to 90% of our biosphere are significantly colonized by cold-adapted organisms. To be able to successfully cope with low temperatures, all these organisms are acclimated on many different molecular levels. One of them concerns a production of cold-active enzymes displaying high catalytic efficiency at low temperatures which is connected with great flexibility and thermosensitivity. Due to their unique characteristics, these biocatalysts have found an enormous potential to participate in various biotechnological processes, such as part of detergents, during degradation of lactose in milk industry, in baking technology and fruit juicy industry, also in the fabric production and treatment, additionally in pharmaceutical and cosmetic industry together with the field of molecular biology and bioremediation.
In this project, we were searching for genes coding for new cold-active enzymes with expected amylase and cellulase activities from various cold-adapted organisms. In the previous work, selected glycosidase activities were tested in different microbial samples adapted to low temperatures. According to the obtained temperature profiles, three strains – two yeast (Mrakiella aquatica and Cryptococcus victoriae) and one bacterial (Arthrobacter sp. C1-1) - were selected showing the biggest ratio of amylase or cellulase activities at low temperatures. To identify genes possibly responsible for the measured activities, we had to design gene-specific (GS) primers first and perform PCR to amplify the wanted coding parts of their genomes. For this purpose, sequences of predicted amylases or cellulases from organisms the most phylogenetically related to the chosen isolates were all downloaded from free available databases and compared. Based on the found similarities – the conserved parts of sequences, the GS primers were devised. The chosen microbial strains were cultivated at 15 °C and used for the isolation of bacterial genomic DNA (gDNA) or total yeast RNA. To amplify bacterial target genes, we performed PCR reactions using the GS primers and the isolated gDNA as a template. In the case of yeasts, cDNA obtained by reverse transcription from the isolated RNA was used as a template to amplify the whole gene sequences by RACE method variations using GS primers combined with special anchor primers.
As for the amylases, we successfully amplified and also sequenced the whole amylase gene from Arthrobacter sp. C1-1 and from Mrakiella aquatica, then sequenced them and analysed by in silico methods. Subsequently, amylase from Arthrobacter sp. C1-1 was recombinantly produced as a fusion protein with polyhistidine-tag in E. coli BL21 (DE3). Its basic temperature, pH and activity parameters were then briefly characterized.
Since we were not able to amplify any cellulase gene from Arthrobacter sp. C1-1 using several designed GS primer pairs, we moved to search for a gene coding for chitinase that could potentially show a cellulase activity too. We succeeded and analysed the found sequence by in silico methods. However, we have not been able to amplify the searched cellulase gene from Cryptococcus victoriae and we are still dealing with it.
In the future, we will complete the identification of yeast cellulase gene and afterwards recombinantly produce, purify and deeply characterize all remaining enzymes.