Metagenomics-based discovery of novel carbohydrate degrading enzymes from deep-sea hydrothermal in situ enrichments; expression, purification and characterization
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- Master theses 
Deep sea hydrothermal vents located along ocean ridges harbor microorganisms with unique adaptations. These extremophiles have adapted to high temperature and extreme conditions. Carbohydrate acting enzymes with thermostable properties are an increasing interest for industrial usage, as their extreme nature enable processes to be performed more efficiently, lower contamination and overall cost. In this study we work with a metagenome that was retrieved from a hydrothermal vent located at Bruse vent site, and then annotated for carbohydrate active enzyme function. From the metagenome 34 sequences were annotated with Carbohydrate Binding Module 9 (CBM9) but lacking enzyme classes. These were further analyzed to determine if they contained signal peptide, transmembrane helices, and two putative xylanases were chosen for further analysis. Two putative xylanases and five annotated α-amylase were placed in pET21a vector, transformed and expressed in E. coli with heterologous protein expression. The target proteins were purified with histidine affinity tag, and enzyme activity was tested through plate assay with xylan and starch substrate. Neopullulanase from a published isolate Geobacillus sp. 12AMOR1 was also purified with histidine affinity tag, and the oligomeric state was estimated through size exclusion chromatography. The putative xylanases were expressed as inclusion bodies, purified under denatured conditions and dialyzed. No activity was observed when tested on plates containing xylan, incubated at 65°C for 4 hours. Two of the annotated α-amylases were expressed as soluble proteins, purified under denatured conditions and dialyzed. No activity was observed when tested on plates containing starch, incubated at 65°C for 4 hours. Based on SDS-PAGE and gel filtration the neopullanase’s oligomeric state was estimated to be a dimer. To achieve active proteins for the putative xylanases we suggest the use of fusion partners to obtain soluble protein, for the α-amylases purification with new histidine tag or ion exchange chromatography to test and confirm the activity of these enzymes.