Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5

Abstract

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s−1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9

“…The K m and V max of EPL15085 toward ulvan were 0.80 ± 0.11 mg·mL –1 and 11.22 ± 0.92 μmol·min –1 ·mL –1 , respectively, and the k cat and k cat / K m were 21.95 ± 0.92 s –1 and 27.44 mL·mg –1, respectively (Table S1 and Figure S4). Compared with those of other reported ulvan lyases (Table ), the V max values of EPL15085 were greater than those of TsUly25B (0.039 ± 0.24 μmol·min –1 ·mL –1 ), ALT3695 (0.11 μmol·min –1 ·mL –1 ), and ULA-3 (2.597 μmol·min –1 ·mL –1 ). ,, In terms of substrate affinity, the K m of EPL15085 was superior to those of PsPL (2.10 ± 0.31 mg·mL –1 ), AsPL (3.19 ± 0.37 mg·mL –1 ), NLR42 (5.1 ± 0.2 mg·mL –1 ), and ULA-3 (1.879 mg·mL –1 ) …”

“…Compared with those of other reported ulvan lyases (Table 2), the V max values of EPL15085 were greater than those of TsUly25B (0.039 ± 0.24 μmol• min −1 •mL −1 ), ALT3695 (0.11 μmol•min −1 •mL −1 ), and ULA-3 (2.597 μmol•min −1 •mL −1 ). 19,29,32 In terms of substrate affinity, the K m of EPL15085 was superior to those of PsPL (2.10 ± 0.31 mg•mL −1 ), 28 AsPL (3.19 ± 0.37 mg•mL −1 ), 16 NLR42 (5.1 ± 0.2 mg•mL −1 ), 13 and ULA-3 (1.879 mg•mL −1 ). 32 3.4.…”

“…The absorbance values were determined at 540 nm . Carbohydrate polyacrylamide gel electrophoresis (C-PAGE) was also employed to detect enzyme activity. − , Ulvan was used at a concentration of 3 g/L in 50 mM Tris-HCl and 5 mM NaCl at pH 8.0 and digested with 0.21 mg/mL EPL15085 at 37 °C overnight. The samples were mixed with an equal volume of a loading buffer .…”

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

“…The K m in this case is significantly lower in comparison to previously characterised enzymes such as ulvan lyases isolated from Thalassomonas sp. LD5 (1.01 mg mL −1 ), Pseudoalteromonas (2.1 mg mL −1 ) and Alteromonas (7.2 mg mL −1 ) (He et al 2017;Qin et al 2018;Wang et al 2022). These differences in kinetic properties could be due to the differences in the ulvan used, as seen in the case of an ulvan lyase extracted for F. agariphila, where the K m of the same enzyme on ulvan extracted from Ulva armonicana, Ulva intestinalis and U. lactuca was 0.7, 3.0 and 1.2 mg mL −1 respectively (Konasani et al 2018).…”

“…These bacteria had no homology to the previously reported ulvan lyases, were aerobic and isolated using conventional media. Several other ulvan lyases from organisms such as Formosa agariphila, Glaciecola sp., Pseudoalteromonas sp., Catenovulum maritimum, and Thalassomonas sp., have been identified and characterised since then and classified into five different CAZy families-PL24, PL25, PL28, PL37 and PL40 (Konasani et al 2018;Qin et al 2018;Li et al 2020;Mondal and Ohnishi 2020;Xu et al 2021;Wang et al 2022;Tang et al 2023). Ulvan lyases are the first enzymes that act on ulvan thereby breaking down the polysaccharide to smaller oligomers and creating a unsaturated end in the process.…”