Proteases from Thermophiles

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

PROTEASES FROM THERMOPHILES AND THEIR BIOTECHNOLOGICAL POTENTIAL... 

Natural thermostable enzymes are expected to be a good model for engineering stabilization of proteins. Study of thermostable enzymes from thermophilic microorganisms has revealed that the hydrophobicity of hydrophobic core inside the protein molecule and the electrostatic interactions of amino acid residues within the folded protein seem to be the cause of their stability. The enzymes from thermophilic microorganisms known so far do not contain the disulfide bond, but aqualysin I. Aqualysin I is an extracellular protease while the others are intracellular enzymes. 

Fig. 10. Sequence alignment of subtilisins S41, SSII, S39, BPN, E, Carlsberg, and thermitase. Thermitase is an homologous subtilisin-like protease from the thermophilic bacterium Thermoactinomyces vulgaris. Residues conserved in four or more of the sequences are shaded. The positions of mutations discovered during the directed evolution of the various subtilisins are indicated above the alignment. E-subtilisin E, F-subtilisin S41, S-subtilisin SSII, B-subtilisin BPN. Active site residues are indicated (A).

Microorganisms are classified into three groups based on their maximum growth temperatures (7) psychrophiles (<30 C), mesophiles (30-55 C), and thermophiles. Thermophiles, which usually grow at temperatures higher than 55 C such as in hot springs, can be classiHed into two subdivisions moderate thermophiles (55-75°C) and extreme thermophiles (>75 C). Most thermostable enzymes are obtained from thermophiles, although some, such as thermostable protease, are from mesophiles. We have purified 20 types of thermostable enzymes from the thermophile. Bacillus stearothermophilus. 

Proteases account for the majority of enzyme sales in industry, the main markets being in the cleaning and food industries, with a small proportion in the chemical and pharmaceutical industries. The scope for extremely (thermo)stable proteases in these commercial niches has been commented on in several reviews [e.g. 278]. Because of the denaturing effect of heat, when mesophilic proteins are substrates for proteases from extreme thermophiles very high specific activities can be attained at high temperatures. Temperature coefficients are also high. 

Thermophilic actinomycetes have been found to be predominant in production of thermophilic proteases. Some proteases of actinomycete origin are highly resistant to heat and denaturing agents. The high thermostability of proteases isolated from thermophilic actinomycete is well... 

Thermophilic protease from fungi have contributed to food industry in a big way. Besides that they have broad substrate specificity and contribute to processing of biomass into fuel (Bazarzhapov et al., 2006 O Donoghue et al., 2008 Macchione et al., 2008 Li et al., 2009 Zanphorlin et al., 2010 Li et al., 2011 Liao et al., 2012 Silva et al., 2014). Broadly, protease enz5me has wide applications in dairy, detergents, pharmaceuticals and food industries. In food industries thermoresistant protease enzymes are widely used as a meat tenderizer, production of amino acid concentrates, etc. 

The present focus is specially on exploring thermophilic proteases for industrial application with special reference to enzyme as part of everyday life. The best studied and most widely used enzymes in the industry are most effective over a temperature range from 40°C to 50°C. At these temperatures, substrate conversion requires long reaction times. One way to overcome these obstacles is to raise the reaction temperature. However, implementing higher reaction temperatures requires the deployment of enzymes that are more thermostable. Therefore, thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels (Berka et al., 2011). The present review focuses specially on exploring thermophilic proteases for industrial application with special reference to TLP production and exploitation for condensation reaction from thermophilic actinomycetes. 

Itoi, Y, Horinaka, M., Tsujimoto, Y, Matsui, H., Watanabe, K. Characteristic features in the structure and collagen-binding ability of a thermophihc collagenolytic protease from the thermophile Geobacillus collagenovorans MO-1. JBacteriol 2006,188( 18), 6572-6579. 

Rai, S. K., Roy, J. K., Mukherjee, A. K. Characterization of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II. Appl Microbiol Biotechnol 2010, 85(5), 1437-1450. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

We isolated the various microorganisms by using conventional isolation techniques from the sea mud. The amount of non-extremophiles was 2.2 x 10 - 2.3 x 10 colonies per gram of dry sea mud (/g of DSM) and about 1% of the isolates from the land. The frequencies of the alkaliphiles and the thermophiles were about 0.67% and 1.7%, almost same as that of the isolates from the land . The barophiles were isolated at the amount of 5-10 colonies /g of DSM. The amylase or protease producers were isolated. The numbers of the amylase producer was... 

Panda, M. K., Sahu, M. K., Tayung, K. Isolation and characterization of a thermophilic Bacillus sp. with protease activity isolated from hot spring of Tarabalo, Odisha, India. IranJ Microbiol im, 5(2), 159-165. 

---