Biochemical Characterization and Cold-Adaption Mechanism of a PL-17 Family Alginate Lyase Aly23 from Marine Bacterium Pseudoalteromonas sp. ASY5 and Its Application for Oligosaccharides Production.

As an important enzyme involved in the marine carbon cycle, alginate lyase has received extensive attention because of its excellent degradation ability on brown algae, which is widely utilized for alginate oligosaccharide preparation or bioethanol production. In comparison with endo-type alginate lyases (PL-5, PL-7, and PL-18 families), limited studies have focused on PL-17 family alginate lyases, especially for those with special characteristics. In this study, a novel PL-17 family alginate lyase, Aly23, was identified and cloned from the marine bacterium Pseudoalteromonas carrageenovora ASY5. Aly23 exhibited maximum activity at 35 °C and retained 48.93% of its highest activity at 4 °C, representing an excellent cold-adaptation property. Comparative molecular dynamics analysis was implemented to explore the structural basis for the cold-adaptation property of Aly23. Aly23 had a high substrate preference for poly ß-D-mannuronate and exhibited both endolytic and exolytic activities; its hydrolysis reaction mainly produced monosaccharides, disaccharides, and trisaccharides. Furthermore, the enzymatic hydrolyzed oligosaccharides displayed good antioxidant activities to reduce ferric and scavenge radicals, such as hydroxyl, ABTS+, and DPPH. Our work demonstrated that Aly23 is a promising cold-adapted biocatalyst for the preparation of natural antioxidants from brown algae.

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

Alginate extracted from widely cultured brown seaweed can be hydrolyzed by alginate lyase to produce alginate oligosaccharides (AOS) with intriguing biological activities. Herein, a novel alginate lyase Aly1281 was cloned from marine bacterium Pseudoalteromonas carrageenovora ASY5 isolated from mangrove soil and found to belong to polysaccharide lyase family 7. Aly1281 exhibited maximum activity at pH 8.0 and 50 °C and have broad substrate specificity for polyguluronate and polymannuronate. Compared with other alginate lyases, Aly1281 exhibited high degradation specificity and mainly produced di-alginate oligosaccharides which displayed good antioxidant function to reduce ferric and scavenge radicals such as hydroxyl, ABTS+ and DPPH. Moreover, the catalytic activity and kinetic performance of Aly1281 were highly improved with the addition of salt, demonstrating a salt-activation property. A putative conformational structural feature of Aly1281 was found by MD simulation analysis for understanding the salt-activation effect.

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

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.

Combined T/Y biliary stent placement for the treatment of biliary obstruction caused by Bismuth-Corlett type IV hilar cholangiocarcinoma.

BACKGROUND/AIMS: To assess the feasibility and efficacy of combined T/Y biliary stent for the bilateral drainage of biliary obstruction caused by Bismuth-Corlett type IV hilar cholangiocarcinoma. METHODOLOGY: Eleven patients with unresectable malignant hilar biliary obstruction of Bismuth-Corlett type IV underwent placement of combined T/Y biliary stent. After unilateral or bilateral percutaneous transhepatic cholangiography, "T" or "Y" type combined biliary metal stents were placed based on the intersection angle of the left and right intrahepatic bile ducts. RESULTS: Technical success of stent placement was achieved for all 11 cases. Eight patients had "T" type and three cases had "Y" type stents placement. Internal drainage achieved with no major complications developed, except for minor hemorrhage occurred for two patients (18.2%). The survival time, liver function including bilirubin concentration and degree of dilation of the bile duct improved. CONCLUSIONS: The combined T/Y biliary stent may be feasible and effective for bilaterally drainage of malignant hilar biliary obstruction, this operation is easy to perform, less invasive and shows a good effect of biliary drainage.