Characterization and overexpression of a novel β -agarase from Thalassomonas agarivorans

Abstract

Aims: The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed. Methods and Results: The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel b-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30Á25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7Á4 and 200 mmol l À1 NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo-and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis. Conclusions:We have successfully cloned and overexpressed the novel b-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided.

Recombinant β-agarases: insights into molecular, biochemical, and physiochemical characteristics

“…The molecular mass of this agarase was between 35 and 100 kDa (Xu et al 2018). A pair of agarases with both exolytic and endolytic activity had been witnessed by Liang et al 2014 andTemuujin et al 2012. A 90 kDa GH50 agarase from a seawater bacterium, Thalassomonas agarivorans BCRC 17492 constituted with 774 amino acids protein along with 26 amino acid signal peptide was observed in this gene and it was designated as AgaB1 (Liang et al 2014).…”

“…A pair of agarases with both exolytic and endolytic activity had been witnessed by Liang et al 2014 andTemuujin et al 2012. A 90 kDa GH50 agarase from a seawater bacterium, Thalassomonas agarivorans BCRC 17492 constituted with 774 amino acids protein along with 26 amino acid signal peptide was observed in this gene and it was designated as AgaB1 (Liang et al 2014). The other agarase was mined from Streptomyces coelicolor A3 (2) and was expressed in Streptomyces lividans TK24 which is a unique host when compared to all other hosts that were used in the expression of the β-agarase gene.…”

“…A few glycoside hydrolases those contain catalytic domains of different GH families are multifunctional enzymes. Depending upon the product profile and specificity to its substrate (Temuujin et al 2012), agarases can be classified into endotype agarase and exotype agarase where they yield neoagarotetraose and neoagarobiose as their end products, respectively (Liang et al 2014). Agarases perform a vital role in the breakdown of agar into simple sugars.…”

“…The recombinant β-agarases belong to the glycoside hydrolase (GH) family such as GH16, GH39, GH50, GH86, and GH118. Predominantly, the recombinant β-agarases were from marine microbes and their genera include Agarivorans (Liu et al 2014a, b;Lee et al 2012;Lin et al 2012), Alteromonas (Seo et al 2014;Chi et al 2014), Aquimarina (Lin et al 2017), Catenovulum (An et al 2018;Cui et al 2014;Xie et al 2013), Cellulophaga (Ramos et al 2018), Flammeovirga (Dong et al 2016;Hou et al 2015;Yang et al 2011;Chen et al 2016;Di et al 2018), Gayadomonas (Lee et al 2018;Jung et al 2017a, b), Microbulbifer (Su et al 2017), Micrococcaceae (Xu et al 2018), Pseudoalteromonas (Chi et al 2015a, b;Oh et al 2010a, b), Pseudomonas (Hsu et al 2015), Saccharophagus (Kim et al 2010(Kim et al , 2017(Kim et al , 2018Lee et al 2013), Simiduia (Tawara et al 2015), Thalassomonas (Liang et al 2014), and Vibrio (Liao et al 2011) whereas few were from soil bacteria Streptomyces (Temuujin et al 2011(Temuujin et al , 2012, Cohnella (Li et al 2015) and one exclusive source was the activated sludges of sewage plant from which Cellvibrio (Osamu et al 2012) had been identified with agarolytic property.…”

Molecular characterization, overexpression and comparison of esterases-encoding LipRT, Lip4 and Lip20 from moderately thermophilic and mesophilic bacteria

“…The bacterial pellet was harvested for genomic extraction. The shotgun library and DNA sequencing were performed as described in Liang et al [5]. Putative open reading frames were further searched and examined using the protein BLAST program available on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).…”