Purification and Characterization of a New Thermostable κ-CarrageenasefromtheMarineBacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KC1 increased its activity slightly. The divalent and trivalent metal ions including Cu^2＋, Ni^2＋, Zn^2＋, Mn^2＋, Al^3＋ and Fe^3＋ significantly inhibited its activity, while Mg^2＋ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.

Purification and characterization of novel κ-carrageenase from marine Tamlana sp.HC4

We isolated a bacterial strain （HC4） that is able to degrade k-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular K-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30~C, respectively. The enzyme is stable over the range ofpH 7.2-8.6 below 45℃. The enzyme activity is strongly inhibited by Zn2＋ and Cu2＋ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant （Kin） at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level ofκ-neocarrabiose sulfate.

Expression and Characterization of a Novelλ-Carrageenase Cgl150A_Wa from Wenyingzhuangia aestuarii

λ-Carrageenan is a highly sulfated polysaccharide alternating of 1,4-O-α-D-galactopyranose-2,6-sulfate(D2S,6S)and 1,3-O-β-D-galactopyranose-2-sulfate(G2S).λ-Carrageenases are desirable tools forλ-carrageenan degradation.Based on the genome mining,a novelλ-carrageenase Cgl150A_Wa was cloned from the bacterium Wenyingzhuangia aestuarii and expressed in Escherichia coli.Cgl150A_Wa was an endo-acting enzyme and exhibited its maximum activity at 30℃and pH 8.0.By employing a glycomics strategy that combined ultra-performance liquid chromatography-mass spectrometry analysis and glycoinformatics,Cgl150A_Wa was proven to degradeλ-carrageenan octaose and hexaose,and the major hydrolysis product of Cgl150A_Wa wasλ-carrageenan tetrose.In addition to the typicalλ-carrageenan motifs,the active center of Cgl150A_Wa might tolerate desulfatedλ-carrageenan motifs.Cgl150A_Wa is a potential biotechnological tool for preparingλ-carrageenan oligosaccharides and structural investigation.

Expanding the application range of theκ‑carrageenase OUC‑FaKC16A when preparing oligosaccharides fromκ‑carrageenan and furcellaran

Carrageenan oligosaccharides are important products that have demonstrated numerous bioactivities useful in the food,medicine,and cosmetics industries.However,the specifc structure–function relationships of carrageenan oligosaccharides are not clearly described due to the defciency of high specifc carrageenases.Here,a truncated mutant OUC-FaKC16Q based on the reportedκ-neocarratetrose(Nκ4)-producingκ-carrageenase OUC-FaKC16A from Flavobacterium algicola was constructed and further studied.After truncating the C-terminal Por_Secre_tail(PorS)domain(responsible for substrate binding),the catalytic efciency and temperature stability decreased to a certain extent.Surprisingly,this truncation also enabled OUC-FaKC16Q to hydrolyze Nκ4 intoκ-neocarrabiose(Nκ2).The ofset of Arg265 residue in OUC-FaKC16Q may explain this change.Moreover,the high catalytic abilities,the main products,and the degradation modes of OUC-FaKC16A and OUC-FaKC16Q toward furcellaran were also demonstrated.Data suggested OUC-FaKC16A and OUC-FaKC16Q could hydrolyze furcellaran to produce mainly the desulfated oligosaccharides DA-G-(DA-G4S)2 and DA-G-DA-G4S,respectively.As a result,the spectrum of products ofκ-carrageenase OUC-FaKC16A has been fully expanded in this study,indicating its promising potential for application in the biomanufacturing of carrageenan oligosaccharides with specifc structures.