Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China; College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China.
Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Int J Biol Macromol. 2020 Jun 1;152:180-188. doi: 10.1016/j.ijbiomac.2020.02.279. Epub 2020 Feb 25.
In this study, a novel pectate lyase (ApPel1) was identified and characterized from Aspergillus parasiticus. The ApPel1 hydrolysed oligogalacturonides (OGs) effectively and produced 4,5-unsaturated OGs from low-methoxyl (LM) pectin, with DP 2 to DP 5 as the major products. Furthermore, the multiple sequence alignments, structure model and phylogenetic analyses of the ApPel1 indicated that its catalytic active sites were highly conserved with other pectin lyases (PLs) and the Ca binding amino acid residues are different compared with pectate lyases (Pels). N187D, N191D and N187D/N191D mutants were constructed to test for both Ca binding properties and the effects on catalytic ability. The three mutations sharply decreased the activity of ApPel1 and Ca tolerance, indicating that the Ca binding amino acid residues are different from the other Pels. Based on the sequence and structure comparison between PLs and Pels, and mutation analysis, the ApPel1 may be direct evolution from PLs. Thus, this enzyme has potential for use in producing unsaturated OGs for biological activity study, and contributes to an improved understanding of the evolutionary relationships between PLs and Pels.
在这项研究中，从寄生曲霉中鉴定并表征了一种新型果胶裂解酶（ApPel1）。ApPel1 能有效地水解低甲氧基（LM）果胶中的寡半乳糖醛酸（OGs），并产生 DP2 到 DP5 为主产物的 4,5-不饱和 OGs。此外，ApPel1 的多重序列比对、结构模型和系统发育分析表明，其催化活性位点与其他果胶裂解酶（PLs）高度保守，与果胶裂解酶（Pels）相比，Ca 结合氨基酸残基不同。构建了 N187D、N191D 和 N187D/N191D 突变体，以测试 Ca 结合特性和对催化能力的影响。这三个突变明显降低了 ApPel1 的活性和 Ca 耐受性，表明 Ca 结合氨基酸残基与其他 Pels 不同。基于 PLs 和 Pels 之间的序列和结构比较以及突变分析，ApPel1 可能是直接从 PLs 进化而来。因此，该酶具有用于生物活性研究中产生不饱和 OGs 的潜力，并有助于更好地理解 PLs 和 Pels 之间的进化关系。
