Enzymes thermophilic protease

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. 

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. 

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. 

Mukherjee, A. K., Adhikari, H., Rai, S. K. (2008). Rroduction of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrical grass and potato peel as low-cost medium Characterization and application of enzyme in detergent formulation. Biochem. Engg. J.,39, 353-361. 

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. 

Aqualysin I is an alkaline serine protease, extracellularly secreted by Thermus aquaticus YT-1,14) an extremely thermophilic, Gram-negative bacterium. The enzyme is thermostable. Optimum temperature for the proteolytic activity of aqualysin I is 80° C in the presence of 1 mM Ca2+ and 70°C in its absence (Fig. 12.1).l5) 85% of the original activity remains after treatment at 80°C for 3 h in the presence of 1 mM Ca2+, but only about 20% remains in its absence.15) These results indicate that calcium ion is essential for the structural stability of the enzyme. 

The high thermostability of natural thermophilic enzymes is usually accompanied by increased resistance to other forms of denaturation, such as cleavage by proteases and chemical denaturation by guanidine hydrochloride or urea. A similar trend is seen in the evolved thermostable esterases. Thermostable 8G8 is more resistant than wild type to cleavage by trypsin and to denaturation by guanidine hydrochloride (Gershenson et al, 2000). 

Thermophilic enzymes (e.g., heat-adapted a-amylase, glucoamylase, cellulase, chitinase, pectinase, p-galactosidase, xylose isomerase, pullulanase, neutral proteases, lipases, and serine and acid proteases) Psychrophilic enzymes (e.g., cold-adapted alcohol dehydrogenase, a-amylase,... 

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. 

Using anaerobic, thermophilic incubation conditions, with wool as the major source of carbon in the growth medium, Riessen and Antranikian [42] isolated a new bacterium that was named Thermoanaerobacterkeratinophilus. This isolate grows optimally at 70°C, and produces an extracellular protease that is responsible for the degradation of native keratin in the wool. One of the goals of this work was to isolate a thermostable keratinolytic enzyme that might be used to modify wool fibers in the textile industry. 

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. 

Thermophiles have a lower biomass yield but an increased product to substrate ratio. They require less incubation time for product formation, higher incubation temperatme and early sporulation, which enhances industrial feasibility. They are known producers of heterologous extracellular enzymes. Among the extracellular enzymes, proteases have high specific activity compared to their mesophilic counterparts. In the global maiket, extracellular enzymes account for two third of the total world maiicet because they are easily extractable and purifiable (Illanes, 1999 Rakshit et al., 2003). Proteases are a unique class of enzymes, which occupy a pivotal position... 

---