A novel thermostable serine protease from a metagenomic library derived from marine sediments in the East China Sea
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
A novel thermostable serine protease from a metagenomic library derived from marine sediments in the East China Sea Jianan Sun 1 & Ping Li 1 & Zhen Liu 1 & Wencan Huang 1 & Xiangzhao Mao 1,2 Received: 9 January 2020 / Revised: 7 August 2020 / Accepted: 2 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Thermal activity and stability are important characteristics for proteases applied in the detergent, pharmaceutical, food, and other green industries. With the intent to discover thermostable novel proteases, we constructed a fosmid metagenomic library from marine sediments in the East China Sea and isolated a clone endowed with high proteolytic activity from this library. Sequence analysis of the positive subclones allowed the identification of a coding region of 1254 bp related to protease activity. The unrooted phylogenetic tree and alignment results revealed that the sequence might be derived from Anaerolineaceae bacterium and encodes a new member of the peptidase S8A subfamily with the typical catalytic triad Asp119/His150/Ser325. The fusion protein, named pF1AL2, was expressed in Escherichia coli and showed a molecular weight of 35 kDa. pF1AL2 was active in the pH range of 5.0–11.0 with an optimal pH at 10.0 and had high stability under alkaline conditions, retaining more than 95% of its activity after 24 h at pH 11.0. The optimal temperature of pF1AL2 was 80 °C, and it retained nearly 80% of its activity after 6 h at 70 °C, showing great thermal activity and stability. In addition, the enzyme had great salt tolerance (the residual activity when kept in 3 M NaCl was 40%). Its thermal activity and stability, along with its halotolerance and pH-tolerance, indicate the high potential value of pF1AL2 in industrial applications. The exploitation of pF1AL2 could lay the foundation for the development and utilization of proteases with special features from marine resources by a metagenomic strategy. Key points • A novel protease, pF1AL2, from marine sediments, was screened out by a metagenomic strategy. • The protease pF1AL2 analyzed in silico, cloned, and characterized. • pF1AL2 had an optimal temperature of 80 °C and retained nearly 80% of activity after 6 h at 70 °C. • pF1AL2 had great tolerance for high-temperature and acid, alkaline, and high salt environments. Keywords Metagenomic library . Marine sediments . Functional screening . Anaerolineaceae bacterium . Serine protease
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10879-3) contains supplementary material, which is available to authorized users. * Xiangzhao Mao [email protected] 1
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
2
Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China
Proteases (hydrolases, EC 3.4.) are enzymes that can hydrolyze peptide bonds and are classified
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