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It has been a long time since the first α-agarase was discovered. However, only two α-agarases have been cloned and partially characterized so far and the study of α-agarases has lagged far behind that of β-agarases. Here, we report an α-agarase, AgaD, cloned from marine bacterium Thalassomonas sp. LD5. Its cDNA consists of 4401 bp, encoding a protein of 1466 amino acids. Based on amino acid similarity, AgaD is classified into glycoside hydrolase (GH) family GH96. The recombinant enzyme gave a molecular weight of about 180 kDa on SDS-PAGE and 360 kDa on Native-PAGE indicating it acted as a dimer. However, the recombinant enzyme is labile and easy to be fractured into series of small active fragments, of which the smallest one is about 70 kDa, matching the size of catalytic module. The enzyme has maximal activity at 35 °C and pH 7.4, and shows a strong dependence on the presence of calcium ions. AgaD degrades agarose to yield agarotetraose as the predominate end product. However, the hydrolysates are rapidly degraded to odd-numbered oligosaccharides under strong alkaline condition. The spectra of ESI-MS and H-NMR proved that the main hydrolysate agarotetraose is degraded into neoagarotriose, bearing the sequence of G-A-G (G, D-galactose; A, 3,6-anhydro-α-L-galactose). Unlike the alkaline condition, the hydrolysates are further hydrolyzed into smaller degree polymerization (DP) of agaro-oligosaccharides (AOS) in dilute strong acid. Therefore, this study provides more insights into the properties for both the α-agarases and the AOS.

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.