Characterization of amylase produced by cold-adapted bacteria from Antarctic samples

Abstract

The Antarctic continent is characterized by extreme conditions and encompasses peculiar environments which represent promising habitats for the recovery of well-adapted microorganisms with unique metabolic capabilities. Enzymatic activity combined to salinity and pH tolerance of bacteria that thrive in low temperature environment have attracted attention of researchers in recent years and, as scarce information on that topic is available, the aim of this study was to prospect bacteria isolated from Antarctic samples for extracellular amylase activity and to determine their salinity and pH tolerances. Ninety-one bacteria isolated from Antarctic environmental samples, including marine sponges and invertebrates, sediments and soil biofilm, were screened. Fourty-two isolates were selected as putative amylase producers based on their ability to produce clearing zones in agar plates. Eighty-six and forty-four isolates were able to grow at 5 and 10% NaCl concentrations, respectively. The best enzymatic activities for isolates 226 and 227 were 768.5 U dL⁻¹ (20 °C) and 1410,16 U dL⁻¹ (20 °C), respectively, both influenced by the temperature. All variables assayed showed statistically significant differences for isolates 227 and 226, demonstrating that enzyme activities respond differently to the applied conditions. This is the first report that describes the amylase activity from Carnobacterium and Psychrobacter strains isolated from Antarctic samples. These results encourage subsequent investigations of enzymes produced by microorganisms adapted to low temperatures and high osmolarity conditions, aiming future biotechnological application in pharmaceutical, textile or food industries.

Introduction

Bioprospecting efforts for the discovery of new secondary metabolites and biocatalysts with novel properties from microorganisms have been increasing in the last years. Industries such as textile, pharmaceutical, food and bioprocessing have great interest in these compounds, increasing the demand for novel and efficient biomolecules. The particularities of the industrial processes encourage the search for substances capable of acting in different conditions and, in this way, the search for new bioactive compounds in peculiar and poorly explored environments, such as Antarctica, may offer an interesting bioprospecting strategy. Some particular properties have been investigated, including the physiological capacity of some microorganisms to tolerate adverse conditions, maintaining their activities under extreme temperatures and/or pH and high salt concentrations (Fulzele et al., 2011).

Oceans cover approximately 71% of the Earth and 90% of the ocean volume is below 5 °C. From the remaining Earth area, 24% is composed of land surface including areas of permafrost (Margesin and Miteva, 2011; Hamdan, 2018). The search for new enzymes produced by psychrophilic and psychrotolerant microorganisms found in Antarctic samples have attracted increasing attention of the scientific community in recent years (Batista et al., 2018), as the microbial communities that live in these environments are adapted to extreme conditions, including low temperatures ranging from −-20 °C to −-60 °C, as well as hypersalinity, low nutrient availability and high UV incidence (Carpenedo et al., 2008; Pascale et al., 2012). Such metabolic plasticity makes the Antarctic a promising environment for the discovery of new microbial cells with unique biochemical properties (Batista et al., 2018).

According to Margesin and Miteva (2011), new genera and species of psychrophilic and psychrotolerant microorganisms have been discovered in different cold habitats, including aquatic and terrestrial environments. Although several studies have shown that there is great biodiversity in the polar regions and the main representative bacterial groups isolated belonged to four major phyla, named Actinobacteria, Bacteroidetes, Proteobacteria, and Firmicutes, the ecological relevance of microbes and their geographical distribution in Antarctic systems is still unknown (Silva et al., 2018).

Presently, the mechanisms of microbial adaptation to environmental conditions have attracted the attention in the search for new enzymes, such as amylases and other stable molecules at low temperatures, which are called extremozymes (Ladygina et al., 2006; Frias et al., 2010; Sarmiento et al., 2015). Amylases are enzymes of great interest for industries, which are mainly related to microorganisms, but that can also be found in organisms such plants and animals (Rao et al., 2007). Enzyme-producing microorganisms adapted to low temperatures offer advantages related to freeze tolerance mechanisms, higher membrane fluidity and production of proteins responsible for adaptation to low temperatures (Kuddus et al., 2012a). Stability of amylases at low temperatures is a highly desirable characteristic, especially for energy saving processes in industries. Scientific interest on microbial cold adapted α-amylases has been increasing due to their extensive application possibilities, including analytical, medicinal and clinical chemistry, as well as starch industry, food processing, detergents, waste-water treatment, biopulping, textile industry, environmental bioremediation in cold climate and other molecular biology applications (Kuddus et al., 2012b).

Due to the importance of cold active enzymes in different fields, the present work aimed to identify psychrotolerant bacteria able to produce cold-active and salt tolerant amylase by enzymatic screening, followed by characterization of the enzymatic activity.