Characterization and Application of a Recombinant Exolytic GH50A β-Agarase from Cellvibrio sp. KY-GH-1 for Enzymatic Production of Neoagarobiose from Agarose

Abstract

Neoagarobiose (NA2) is the repeating disaccharide unit of agarose and possesses various promising biological activities. To identify an efficient exolytic βagarase required for NA2 production from agarose, the GH50A β-agarase gene from agar-degrading Cellvibrio sp. KY-GH-1 was overexpressed as a recombinant Histagged protein using the Escherichia coli expression system. GH50A β-agarase that consists of 797 amino acids was able to produce predominantly NA2 from agarose at an optimal temperature and pH of 35 °C and 7.5, respectively. The enzyme was stable up to 35 °C and within a pH range of 7.0−9.0. The K m , V max , K cat , and K cat /K m values of the enzyme were 26.5 mg/mL, 16.9 U/mg, 25.2 s −1 , and 1.2 × 10 5 s −1 M −1 , respectively. The copresence of 5 mM MnSO 4 and 10 mM tris(2-carboxyethyl)phosphine (TCEP) resulted in a 2.5-fold enhancement of the enzyme activity. For NA2 production, neoagaro-oligosaccharides (NAOSs) containing NA4−NA18 were preferred over agarose or agaro-oligosaccharides (AOSs) as substrates. NA2 was produced along with minor amounts of agarotriose (A3) after treatment of AOS with the enzyme, indicating that the exolytic digestion of AOS by the enzyme was initiated by releasing A3 from nonreducing ends. Enzymatic hydrolysis of 0.4% agarose (100 mL) using GH50A β-agarase (20 μg/mL) for 4 h under optimal reaction conditions (5 mM MnSO 4 , 10 mM TCEP, 35 °C, 20 mM Tris−HCl, and pH 7.5) and purification of NA2 from hydrolysis products by Bio-Gel P-2 column chromatography resulted in the recovery of 216 mg of NA2 (∼54% yield from agarose). Altogether, these results suggest that the recombinant GH50A β-agarase is useful to convert agarose to NA2.

Extracellular production of a thermostable Cellvibrio endolytic β-agarase in Escherichia coli for agarose liquefaction

“…A standard NAOS mixture (NA2–NA18) was provided by Dr. Sang-Hyeon Lee (Lee et al 2008 ). An AOS mixture was prepared by mild acid hydrolysis of 1%[w/v] agarose as previously described (Kwon et al 2020 ). The standards (NA2, NA4, and NA6) were obtained from Carbosynth Ltd. (Berkshire, UK).…”

“…Endo-acting β-agarases from the GH16, GH86, and GH118 families degrade agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6), NA6 and neoagarooctaose (NA8), and NA8 and neoagarodecaose (NA10), respectively. The β-agarase members of GH50 family degrade agarose via exo-acting activity or a combination of exo- and endo-acting activities to produce neoagarobiose (NA2) (Kwon et al 2020 ; Han et al 2016 ), NA4 (Chen et al 2018 ), or NA2 and NA4 (Li et al 2015 ; Liang et al 2017 ) as end products. GH96 α-agarases hydrolyze agarose into agarotetraose (Flament et al 2007 ; Lee et al 2019 ), whereas GH117 α-neoagarobiose hydrolases (α-NABH) cleave NA2 into L-AHG and D-Gal (Ha et al 2011 ; Jang et al 2021 ).…”

Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

“…In detail, 10 μL enzyme solution was mixed with 240 μL agarose (0.4%; w / v ) with different pHs including 3.0–8.0 (50 mM sodium citrate-dibasic sodium phosphate), 8.0–9.0 (50 mM Tris-HCl), and 9.0–10.6 (50 mM glycine-NaOH), and the mixture was incubated at the temperatures of 0, 10, 20, 30, 35, 40, 45, 50, 60, 70, and 80 °C for 10 min, respectively. Then, 750 μL 3,5-dinitrosalicylic acid (DNS) solution was added, and the resulting sugar was measured by the DNS method [ 28 ]. To determine the stability against pH, rAgaW1540 was pre-incubated in the above-mentioned pH buffers at 25 °C for 60 min, and then the residual activity was determined.…”

Simultaneous quantification of marine neutral neoagaro-oligosaccharides and agar-oligosaccharides by the UHPLC-MS/MS method: application to the intestinal transport study by using the Caco-2 cell monolayer

“…11 These activities are likely related to the presence of AHG and DP in the structure. 12,13 In fact, oligosaccharides prepared from agarose have a wide range of applications in the pharmaceutical, food, and cosmetic industries. 14,15 However, the intestinal transport study of NAOS and AOS with different DP in vitro has not been reported.…”

“…To characterize and quantify, a procedure for simultaneous determination of NAOS and AOS with different DP was required. Currently, a variety of analytical methods for components of NAOS and AOS have been published, including HPLC-ELSD, 12 MALDI-TOF/MS, 22,23 HPAEC-PAD, 24,25 LC-MS/MS, 13 and LC/MS-IT-TOF 26 (Table 1). However, most of them were qualitative analysis used for the separation and purication of crude products.…”