Molecular cloning and characterization of a halotolerant α-amylase from marine metagenomic library derived from Arabian Sea sediments.

Functional screening of a metagenomic library of marine sediment revealed an amylolytic clone BTM109. This report states the purification and characterization of a moderately halotolerant α-amylase, with more than 51% activity in 2.5 M NaCl. The molecular mass of purified protein was determined to be 55.7 kDa by MALDI-TOF MS. The optimum pH for enzyme activity was pH 7 and temperature for maximal activity was 40 °C. At 5 mM concentration, Ca2+ enhanced the enzyme activity indicating that the enzyme is a Ca2+ dependent α-amylase which was confirmed by the starch hydrolysis pattern using TLC. These physico-chemical properties support the suitability of this enzyme for various industrial applications.

Evaluation of five in situ lysis protocols for PCR amenable metagenomic DNA from mangrove soils.

Microbes in nature are rarely amenable to growth by standard microbiological methods, with the majority being unculturable. Metagenomic methods help to bypass and overcome the limitations of traditional culturing method; wherein total community DNA is isolated, cloned into suitable vector and host systems. However, isolation of total community DNA itself remains a challenge. In this study five methods of total community DNA isolation from three different mangrove soils were evaluated to test its PCR amenability. The yield and purity of the isolated DNA was also analysed. The quantity of DNA by all 5 methods although reasonably high, contained residual humic contaminants. Of the five, the method employing liquid nitrogen yielded readily amplifiable DNA, while that by all others required further downstream processing to achieve purity and PCR amenability.