Cloning, expression, and characterization of a new pH‐ and heat‐stable alginate lyase from Pseudoalteromonas carrageenovora ASY5

Abstract

Alginate lyase is important in marine alginate degradation, and its enzymatic hydrolysates are excellent antioxidants. Here, we cloned a new alginate lyase, that is, Alg823, from the Gram‐negative marine bacterium Pseudoalteromonas carrageenovora ASY5. The optimal temperature and pH of Alg823 were 55°C and pH 8.0, respectively. After 30 min of incubation at 50°C, Alg823 could maintain over 75.0% of the maximum enzyme activity, suggesting its thermostability. The recombinant alginate lyase retained more than 80.0% of the maximum enzyme activity after it was treated at pH 6.0–10.0 and 4°C for 24 hr, indicating its excellent pH stability. Mg2+, Ca2+, Na+, and K+ could promote enzyme activity. Alginate oligosaccharides obtained by degradation with Alg823 displayed an excellent ability to scavenge ABTS, hydroxyl, and DPPH radicals. Alg823 showed potential for novel applications in alginate oligosaccharide production because of its pH tolerance and heat adaptation. Practical applications Alginate oligosaccharides produced by alginate degradation possess favorable properties, such as low molecular weight, high stability, and co‐dissolution with water. These oligosaccharides also have many biological activities. As such, they have been widely explored. Alginate oligosaccharides are prepared via three methods, namely, physical, chemical, and enzymatic methods. In chemical method, operational processes are difficult to thereby possibly damaging the unique structure of polysaccharides and causing environmental pollution. Although physical methods can overcome some of the shortcomings of chemical methods, their reaction is still difficult to control, and products are complicated. Conversely, enzymatic methods can has advantages of mild conditions, single product, and less pollution. Furthermore, oligosaccharides prepared by enzymatic methods are more biologically active than those prepared by other methods. Thus, finding novel alginate lyase with high activity and stability is important for research and commercial purposes.

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5

“…At the temperature range of 45-55 °C, Aly1281 manifested over 50% of the activity observed at the optimal temperature. Few studies had reported alginate lyases that exhibited optimal reaction temperatures higher than 50 °C, such as AMOR_PL7A (65 °C) [22], cAlyM (55 °C) [23] and Alg823 (55 °C) [5]. The thermostability of Aly1281 was determined at a temperature ranging from 0 °C to 70 °C.…”

“…Aly1281 exhibited maximum enzyme activity at 50 • C. At the temperature range of 45-55 • C, Aly1281 manifested over 50% of the activity observed at the optimal temperature. Few studies had reported alginate lyases that exhibited optimal reaction temperatures higher than 50 • C, such as AMOR_PL7A (65 • C) [22], cAlyM (55 • C) [23] and Alg823 (55 • C) [5]. The thermostability of Aly1281 was determined at a temperature ranging from 0 • C to 70 • C. As shown in Figure 3B, Aly1281 was relatively stable at temperatures lower than 55 • C. Aly1281 showed competitive thermostability in comparison with most reported marine-derived alginate lyases [15,[24][25][26], which could benefit its industrial application.…”

“…Hydroxyl radical scavenging activity was determined in accordance with a previously described method [5]. Briefly, 0.1 mL of FeSO 4 (9.0 mM), 0.6 mL of deionized water, 2 mL of oligosaccharide solution, 0.1 mL of H 2 O 2 (8.8 mM), and 0.1 mL of ethanol salicylate (9.0 mM) were mixed and incubated at 37 • C for 30 min.…”

“…Due to the widespread cultivation of brown seaweed in Asia, alginate has been extensively studied for its extraction ability and is widely used as a viscosifier, stabilizer, and gelling agent in the food [2], chemical [3] and biomaterials [4] industries. Many recent studies have demonstrated that alginate oligosaccharides (AOS), which exhibit outstanding biological properties that differ from those of alginate polysaccharides, can be produced by further hydrolysis of alginate; in particular, AOS presents anti-antioxidant [5], anticoagulant [6] and antineoplastic [7] properties. Some studies have shown that AOS can lower blood sugar and blood lipid levels [8] and display anti-tumor [9] and anti-viral [10] abilities.…”

Characterization of a New Bifunctional and Cold-Adapted Polysaccharide Lyase (PL) Family 7 Alginate Lyase from Flavobacterium sp.

“…However, few of these reported enzymes have been commercially used owing to their poor properties [ 56 ]. Several alginate lyases with certain specific characteristics have been found and studied, such as pH stability [ 51 , 57 ], thermo-tolerance [ 58 ], cold adaption [ 59 , 60 ], acidic or alkaline adaption [ 61 , 62 ], single product distribution [ 63 , 64 ], which are quite significant for both research and commercial purposes, but the enzymes with optimal characteristics are rare. It is worth noting that cold-adapted alginate lyases make running biocatalysis at low temperature possible, which is crucial to reduce the danger of contamination and improve the sustainability of enzyme utilization, thereby decreasing the input costs to some extent, particularly for industrial production of oligosaccharides.…”