Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber

Abstract

A novel endo-type β-agarase was cloned from an agar-degrading bacterium, Microbulbifer sp. Q7 (CGMCC No. 14061), that was isolated from sea cucumber gut. The agarase-encoding gene, ID2563, consisted of 1800 bp that encoded a 599-residue protein with a signal peptide of 19 amino acids. Sequence analysis suggested that the agarase belongs to the GH16 family. The agarase was expressed in Escherichia coli with a total activity of 4.99 U/mL in fermentation medium. The extracellular enzyme activity accounted for 65.73% of the total activity, which indicated that the agarase can be extracellularly secreted using the wild-type signal peptide from Microbulbifer sp. Q7. The agarase exhibited maximal activity at approximately 40 °C and pH 6.0. It was stable between pH 6.0 and pH 9.0, which was a much wider range than most of the reported agarases. The agarase was sensitive to some metal ions (Cu 2+ , Zn 2+ and Fe 3+), but was resistant to urea and SDS. The agarase hydrolyzed β-1,4-glycosidic linkages of agarose, primarily yielding neoagarotetraose and neoagarohexaose as the final products. These indicate that this recombinant agarase can be an effective tool for the preparing functional neoagaro-oligosaccharides.

Evaluation of Prebiotic Potential of Three Marine Algae Oligosaccharides from Enzymatic Hydrolysis

“…Alginate lyase and agarase were both produced from marine bacterium Microbulbifer sp. Q7 [57,58], and κ-carrageenase was produced from Zobellia sp. ZM-2 [59].…”

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

“…The protein denaturant urea contradictorily did not affect the agarases ID2563, YM01-1, and YM01-3 (Rajagopalan et al 1961). In point of fact, the agarase ID2563 was resistant to urea and the agarase activity of YM01-1 and YM01-3 was not affected by urea (Su et al 2017;An et al 2018;Cui et al 2014). However, the presence of urea negatively influenced the agarase AgaM1 (Di et al 2018).…”

“…In the study conducted by Hou et al (2015), where Gracilaria lemaneiformis was used as the substrate for the recombinant agarase AgaP4383 exhibited a V max of 21 U/ mg and a K M of 32.41 mg/ml where it produced NA4 and NA6 as the products. Many other different agarases such as AgaB, ID2563, Aga16B, Aga4436, AgaH92, AgaA, Aga2, YM01-3, Agy1, DagA, AgaM1, and AgrP also yielded NA4 and NA6 as the degradation product from the substrate agarose, and exceptionally, they were obtained as products by the action of AgrP on agar (Dong et al 2016, Su et al 2017Kim et al 2017;Chen et al 2016;Chi et al 2015a, b;Hsu et al 2015;Ramos et al 2018;Cui et al 2014;Lee et al 2013;Temuujin et al 2011;Di et al 2018;Oh et al 2010). Of all the agarases, the K M of this agarase, AgaP4383, was found to be higher which makes us understand that the binding affinity of this agarase with the substrate is lesser, as the substrate is not directly accessible to the enzyme to act on it.…”

“…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.…”

Complete Genome Sequence of Cellulase-Producing Microbulbifer sp. Strain GL-2, Isolated from Marine Fish Intestine

“…The genus Microbulbifer has been reported from salty environments such as seawater ( 1 ), marine sediment ( 2 ), mangrove forest ( 3 ), marine algae ( 4 ), and sea cucumber gut ( 5 ). It is known that Microbulbifer isolates from mangrove sediment can degrade a variety of polysaccharides, including cellulose, and the whole genome has been reported ( 6 ).…”