Expression and Characterization of a Novel λ-Carrageenase Cgl150A_Wa from Wenyingzhuangia aestuarii

Characterization of a λ-Carrageenase Mutant with the Generation of Long-Chain λ-Neocarrageenan Oligosaccharides

“…However, the λ-carrageenases from Pseudoalteromonas carrageenovora and Wenyingzhuangia aestuarii performed better when it was 35 °C and 30 °C, while the Cga-L50 from Bacillus sp. had a better activity at 75 °C [ 17 , 18 , 20 , 22 ]. Its temperature stability had decreased dramatically compared to OUC-CglA, but it was more stable at 15 °C and 10 °C [ 20 ].…”

“…CL19 was 7.5 and 7.0, while the Cga-L50 from Bacillus sp. and the Cgl150A_Wa from Wenyingzhuangia aestuarii had an optimal pH of 8.0 [ 17 , 18 , 20 , 21 , 22 ]. Below pH 6.0 and above pH 8.0, the activity significantly decreased and was less than 20% of that at pH 7.0 ( Figure 3 c).…”

“…The product identification by HPLC-MS revealed that the final products of the mutant were mostly long-chain oligosaccharides, including λ-neocarradecasaccharide (Nλ10), λ-neocarradodecasaccharide (Nλ12), λ-neocarratetradecasaccharide (Nλ14), λ-neocarrahexadecasaccharide (Nλ16), λ-neocarraoctadecasaccharide (Nλ18), and λ-neocarraeicosaccharide (Nλ20) ( Figure 4 c,d), while those of OUC-CglA were mainly oligosaccharides with lower polymerization degree such as Nλ2, Nλ4 and Nλ6 [ 20 ]. The ultimate products of the reported λ-carrageenases are currently mostly Nλ2, Nλ4 and Nλ6 [ 17 , 18 , 19 , 20 , 21 , 22 ]. Nevertheless, the ultimate products of the mutant λ-carrageenase OUC-CglA-DPQQ without the PQQ-like domain were notably distinct from those produced by all the other λ-carrageenases described, revealing it to be a promising tool for the preparation of long-chain oligosaccharides and suggesting that the PQQ-like domain may be part of the catalytic groove and related to the minimal substrate that the groove can bind [ 32 ].…”

“…Some progress has been made in the enzymatic degradation of λ-carrageenan. Several λ-carrageenases have been found and characterized, and the ultimate products of these λ-carrageenases which have been reported until now, mostly consist of λ-neocarrabiose (Nλ2), λ-neocarratetraose (Nλ4) and λ-neocarrahexaose (Nλ6), and they are all short-chain oligosaccharides [ 17 , 18 , 19 , 20 , 21 , 22 ]. Therefore, more research studies into the preparation of long-chain λ-neocarrageenan oligosaccharides are essential.…”

“…In this study, the PQQ-like domain was excised and its effects were explored. It was discovered that the majority of final products of the mutant OUC-CglA-DPQQ were longchain oligosaccharides, which had a difference compared to that of other λ-carrageenases reported [17][18][19][20][21][22], suggesting that the mutant could be a candidate enzyme to produce longer-chain λ-neocarrageenan oligosaccharides. Additionally, without the PQQ-like domain, fewer inclusion bodies were produced throughout the expression process and the yield of the λ-carrageenase increased about five-fold.…”