Khizhnyak S.V. , Pampuha V.T., Kovalenko A.Y.

Cold-adapted amylase producers from karst cave "Karaulnaya-2"

Krasnoyarsk State Agrarian University

Amylases are one of the main enzymes used in industry and are occupying approximately 25% of the world enzyme market (De Souza, 2010). In recent yeas it is observed the growing interest to the psychrophilic and psychrotolerant producers of cold-active amylases (Kuddus et al., 2011; Sarmiento et al., 2015). Up to now the main sources of such producers were Antarctica, Arctic, high-mountain and deep-water ecosystems. Our researches demonstrated that cold limestone caves are the natural source of the psychrophilic and psychrotolerant bacteria and fungi (Khizhnyak et al., 2003). The present work is devoted to assessment of caves as an alternative source of cold-adapted amylase producers.

Two strains of cold-adapted amylolytic fungi of the genus Geomyces isolated from low-temperature karst cave "Karaulnaya-2" were examined for their growth rates at different temperatures in the range of +8 to +30°C and for the effect of temperature on amylolytic activity of their cultural liquids (CL). Effect of temperature on growth rates of both strains is well described by Ratkowsky et al. (Ratkowsky et al., 1983) equation with the coefficients of determination R² = 0.987 for the psychrophilic strain and R² = 0.997 for the psychrotolerant strain, theoretical optimum temperatures are +18.5 and +24.5°C, respectively (fig. 1).

Thet effect of temperature on amylolytic activity of CL in the range of +31 to +50°C is well described by the logistic function (R² = 0.994 for the psychrophilic strain and 0.997 for the psychrotolerant strain). The temperature value of the sigmoid's midpoint for the psychrophilic strain is 7°C lower then that of the psychrotolerant strain, what is almost equal to the difference in the optimal growth temperatures of these strains (6°C). The slope of log-lin plot of amylolytic activity of CL of the psychrophilic strain in the exponential area of the temperature curve is 2 times higher than that of the psychrotolerant strain (fig. 2).

Fig 1. Effect of temperature on the hyphal apex growth rate of the psychrophilic and psychrotolerant strains: Ps-E – experimental data for the psychrophilic strain, Pt-E – experimental data for the psychrotolerant strain, Ps-T – the least squares fit of Ratkowsky's equation for the psychrophilic strain, Pt-T – the least squares fit of Ratkowsky's equation for the psychrotolerant strain.

Fig 2. Log-lin plots of amylolytic activity of CL of the psychrophilic and the psychrotolerant strain in the exponential area of the temperature curve.

These results show that decrease of optimal growth temperature in the process of adaptation to cave environment leads to the corresponding decrease of the temperature optimum of amylolytic enzymes and demonstrate perspectives of search for low-temperature amylase producers in cave communities.

References

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2. Khizhnyak, S.V., Tausheva, I.V., Berezikova, A.A., Nesterenko, Y.V., Rogozin D.Y. Psychrophilic and Psychrotolerant Heterotrophic Microorganisms of Middle Siberian Karst Cavities. Russian Journal of Ecology, 2003, 34 (4): 231-235.

3. Kuddus, M., Roohi, J.M.A., Ramteke P.W. An overview of cold-active microbial α-amylase: Adaptation strategies and biotechnological potentials. Biotechnology, 2011, 10: 246–258.

4. Ratkowsky, D.A., Lowry, R.K., McMeekin, T.A., Stokes, A.N., Chandler, R.E. Model for bacterial culture growth rate throughout the entire biokinetic temperature range. J. Bacteriol., 1983, 154: 1222–1226.

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