Isolation and characterization of thermophilic bacteria from Omani hot springs and heterologous expression and purification of potential thermostable enzymes.

Microorganisms can thrive in all possible environments including extreme habitats such as northern and southern polar areas, high saline zones and hydrothermal vent systems. Of particular interest for industrial biotechnology are thermophiles, microorganisms which grow and survive at elevated temperatures, since they act as indispensable sources of thermostable enzymes. Thermostable enzymes such as cellulases, amylases, lipases, proteinases and chitinases have many desirable characteristics which make them attractive targets for a wide range of industrial applications. Commercial applications of enzymes include hydrolyzing biopolymers into smaller components, leather preparation, detergents, diagnostics and food processing. Hot springs are considered favorable habitats for thermophilic microorganisms which can produce novel enzymes with unique properties awaiting to be discovered. Thermostable enzymes offer advantages over other enzymes because they remain active at high temperatures, tolerate proteolysis and severe conditions. The Sultanate of Oman has several natural hot springs which have unique biological characteristics making them attractive subjects for research with potential value for industrial biotechnology purposes. No reports about the isolation of thermophilic bacteria from hot spring in Oman has been published yet and little is known about the occurrence and distribution of thermophiles in the Sultanate. The aim of the present proposal is to isolate, identify and characterize thermophilic microbial diversity from several hot springs in Oman by combining various phenotypic (morphology, microscopic and biochemical) and genotypic methods. Furthermore, a number of enzymatic activities (cellulase, lipase, amylase and proteinase) will be screened from samples collected from hot springs. The potential enzymes will be produced in the native bacteria followed by purification ammonium sulphate purification method and their application in different commercial fields will be evaluated. To maximize the yields to commercial levels, the potential enzymes will be recombinantly overexpressed as fusion proteins with GFP in heterologous established protein expression systems (E. coli and/or L. lactis). The levels of protein expression will be assessed by checking fluorescence of the fusion protein under fluorescent microscopy and the possibility of expressing the protein in large scale will be evaluated.

Microorganisms can thrive in all possible environments including extreme habitats such as northern and southern polar areas, high saline zones and hydrothermal vent systems. Of particular interest for industrial biotechnology are thermophiles, microorganisms which grow and survive at elevated temperatures, since they act as indispensable sources of thermostable enzymes. Thermostable enzymes such as cellulases, amylases, lipases, proteinases and chitinases have many desirable characteristics which make them attractive targets for a wide range of industrial applications. Commercial applications of enzymes include hydrolyzing biopolymers into smaller components, leather preparation, detergents, diagnostics and food processing. Hot springs are considered favorable habitats for thermophilic microorganisms which can produce novel enzymes with unique properties awaiting to be discovered. Thermostable enzymes offer advantages over other enzymes because they remain active at high temperatures, tolerate proteolysis and severe conditions. The Sultanate of Oman has several natural hot springs which have unique biological characteristics making them attractive subjects for research with potential value for industrial biotechnology purposes. No reports about the isolation of thermophilic bacteria from hot spring in Oman has been published yet and little is known about the occurrence and distribution of thermophiles in the Sultanate. The aim of the present proposal is to isolate, identify and characterize thermophilic microbial diversity from several hot springs in Oman by combining various phenotypic (morphology, microscopic and biochemical) and genotypic methods. Furthermore, a number of enzymatic activities (cellulase, lipase, amylase and proteinase) will be screened from samples collected from hot springs. The potential enzymes will be produced in the native bacteria followed by purification ammonium sulphate purification method and their application in different commercial fields will be evaluated. To maximize the yields to commercial levels, the potential enzymes will be recombinantly overexpressed as fusion proteins with GFP in heterologous established protein expression systems (E. coli and/or L. lactis). The levels of protein expression will be assessed by checking fluorescence of the fusion protein under fluorescent microscopy and the possibility of expressing the protein in large scale will be evaluated.

Enzymes that have been characterized from thermophilic microorganisms include amylases, cellulases, chitinases, xylanases, pectinases, proteases, lipases, and esterases. There are novel biotechnological applications of thermophilic enzymes in several industries such as agriculture, pharmaceutical, detergent, textile, leather, pulp and paper, food beverages and feed, and biomining 4. For example, cellulase enzymes have shown their potential applications in the production of bioethanol and value-added organic compounds for renewable agricultural residues and production of glucose feedstock from agricultural cellulosic supply 5. In addition, cellulases play a crucial role in the bleaching pf paper and in environmental bioremediation 1. Proteases are used as harmless additives in commercial detergents and in synthesis of bioactive peptide. Proteases constitute around 65% of total enzyme market and is considered one of the most commercialized enzyme 6. Amylases function in the hydrolysis of starch to maltose and glucose, and they are utilized in a wide variety of nutritional, cosmetic and pharmaceutical industries 7. A special area of interest that has arisen with the increasing concern on the possible depletion of fossil fuel resources along with the environmental issues is the conversion of biomass into sugars by bio-catalytic reactions. The aforementioned process, known as biorefining, agricultur8al crops and other renewable resources are exploited for either extraction of intermediates or for direct bioconversion into fuels and chemicals. The catalysts used in these processes are preferably thermostable enzymes as high temperatures play an important role in stimulating the disintegration of raw materials as well as enzyme penetration 8.The novel thermostable enzymes/bioproducts will potentially be produced in sufficient amounts with the aligned advancement in molecular biology tools 8. As stated in the Business Company Research report (BCC Jan, 2017), the global market for industrial enzymes was approximately $4.9 billion in 2015 and is expected to increase to $6.3 billion by 20219. Hot springs represent a unique diversity of thermophilic species that produce a variety of useful molecules including biocatalysts with high thermal stability suitable for agricultural, industrial and medical processes10. Thermal extreme environments around the world have been a source of several novel thermophilic microbes such as Herbinix luporum, Thermoanaerobabcter uzonensis and Thermotoga elfii11.