This study presents a simple method to immobilize β-agarase onto amino-modified magnetic nanoparticles (MNPs) by biotin/streptavidin (BT/SA) recognition. The effect of BT functionalized with different lengths of poly(ethylene glycol) (PEG) on the activity and stability of heterogeneous biocatalysts was also investigated. The results showed that the enzymatic activity was retained above 93% under the immobilization conditions (pH 7.0, 25 °C, 6 h, and β-agarase concentration of 1 mg/mL). The optimal pH and temperature for both free and immobilized β-agarases were 7 and 30 °C, respectively. PEG length played an important role in the catalytic performance of the immobilized enzyme. As the PEG length increased, an increased trend in activity retention and yield of neoagarobiose was observed. Ultimately, the introduction of PEG24 improved the activity retention of immobilized β-agarase up to 135.67%, and retained 84.18 and 37.04% of residual activity after incubation for 6 h at 40 and 45 °C, respectively, while only 30.50 and 0% of residual activities were observed for free β-agarase at these temperatures, indicating the significantly enhanced stability of the β-agarase after immobilization. Additionally, β-agarase-BTPEG24-SA@MNPs showed the highest yield of neoagarobiose at 45 °C, which was 1.89-fold higher than that of the free enzyme. These results indicated that immobilization of β-agarase on BTPEG-SA@MNPs improved both the catalytic efficiency and stability of the enzyme, thereby promoting the continuous production of neoagarobiose.
Agarose, a hydrophilic polysaccharide derived from red algae, is widely used in biochemical and agricultural applications, but its intrinsic gelation below ~40 °C limits enzymatic conversion into high-value neoagarooligosaccharides (NAOS). Here, we report the systematic characterization of a novel thermophilic β-agarase, Sa AgaD, identified from Salegentibacter agarivorans . Sa AgaD is a 398-residue GH16 enzyme that expresses solubly in Escherichia coli and exhibits a high catalytic efficiency ( k cat / K m = 1.76 × 10 4 mM −1 s −1 ) toward agarose. Distinct from many characterized agarases, Sa AgaD displays optimal activity at pH 8.0 and 55 °C, retains ~90% activity at 65 °C, and remains stable under neutral to mildly alkaline conditions, enabling effective hydrolysis above the agarose gelation point. The enzyme preferentially degrades agar and crude Gracilaria agar, with minimal activity on sulfated porphyran. Product profiling by HPAEC-PAD and ESI-MS demonstrates an endolytic mechanism yielding predominantly neoagarotetraose (NA4) and neoagarohexaose (NA6), with a scale-up NAOS yield of ~75%. Molecular dynamics simulations of Sa AgaD and its NA8 complex reveal a stable substrate binding mode and highlight flexible loop regions that coordinate substrate recognition. The combination of high thermal tolerance, catalytic efficiency, and product specificity positions Sa AgaD as a promising biocatalyst for industrial NAOS production from marine biomass.
Xuewu Liu,Xingfei Li,Zhengjun Xie,Xing Zhou,Long Chen,Chao Qiu,Cheng Lu,Zhengyu Jin,Jie Long
Agarose from biomass can be used to synthesize the rare sugar 3,6-anhydro- L -galactose ( L -AHG), and the new synthesis route and functional properties of L -AHG have always been the focus of research. Here we developed a novel method to co-immobilize Aga50D and Bp GH117 onto streptavidin-coated magnetic nanoparticles and achieved the conversion of agarose to bioactive L -AHG in one pot. Results showed that enzymes were successfully immobilized on the carrier. The activity of co-immobilized enzymes was 2.5-fold higher than that of single immobilized enzymes. Compared with free enzymes, co-immobilized enzymes exhibited enhanced thermal stability. The co-immobilized enzymes retained 79.45 % relative activity at 40 °C for 3 h, while the free enzymes only possessed 21.40 % residual activity. After eight cycles, the co-immobilized enzymes still retained 73.47 % of the initial activity. After silica gel chromatography, the purity of L -AHG obtained by co-immobilized enzymes hydrolysis reached 83.02 %. Furthermore, bioactivity experiments demonstrated that L -AHG displayed better antioxidant and antibacterial effects than neoagarobiose. L -AHG had broad-spectrum antibacterial activity, while neoagarobiose and D -galactose did not show an obvious antibacterial effect. This study provides a feasible method for the production of L -AHG by a co-immobilized multi-enzyme system and confirms that L -AHG plays a key role in the bioactivity of neoagarobiose.
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