Liu Xiaohua, Xiao Zhongbin, Li Kuikui, Wang Wenxia, Jia Xiaochen, Li Tang, Yin Heng
Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Dalian Technology Innovation Center for Green Agriculture, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Dalian Technology Innovation Center for Green Agriculture, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Int J Biol Macromol. 2025 Jan;285:138304. doi: 10.1016/j.ijbiomac.2024.138304. Epub 2024 Dec 2.
Alginate lyases, which degrade alginate into oligosaccharides, have broad applications in biorefinery, biomedical, and industrial fields. The Polysaccharide Lyase Family 7 (PL7) is particularly notable for its alginate lyase activity. In this study, two novel alginate lyases, PmAlg7A and PmAlg7B, from Pseudomonas mendocina E03 were cloned, heterologously expressed, and characterized. PmAlg7B exhibited limited activity toward alginate (0.10 U/mg-protein), while PmAlg7A demonstrated higher activity with a specific activity of 0.76 U/mg-protein. PmAlg7A was identified as an MG-specific alginate lyase, producing oligosaccharides with degrees of polymerization (Dp) ranging from 2 to 5. The enzyme exhibited optimal activity at a temperature of 30 °C and a pH of 8.0, with a K of 7.94 ± 0.92 mg/ml and a k of 1.23 ± 0.06 s. Structural comparisons and amino acid sequence alignments indicated a potential role for residue 55 in loop B in modulating the activity of PmAlg7B, which was supported by mutagenesis experiments and molecular dynamics simulations. These findings enhance our understanding of the critical role of loop B in regulating substrate binding in PL7 alginate lyases.
能够将藻酸盐降解为寡糖的藻酸盐裂解酶在生物精炼、生物医学和工业领域有着广泛的应用。多糖裂解酶家族7（PL7）因其藻酸盐裂解酶活性而格外引人注目。在本研究中，从门多萨假单胞菌E03中克隆、异源表达并表征了两种新型藻酸盐裂解酶PmAlg7A和PmAlg7B。PmAlg7B对藻酸盐表现出有限的活性（0.10 U/mg蛋白），而PmAlg7A表现出更高的活性，比活性为0.76 U/mg蛋白。PmAlg7A被鉴定为一种MG特异性藻酸盐裂解酶，可产生聚合度（Dp）为2至5的寡糖。该酶在30℃温度和8.0 pH下表现出最佳活性，K为7.94±0.92 mg/ml，k为1.23±0.06 s。结构比较和氨基酸序列比对表明，环B中的55位残基在调节PmAlg7B活性方面具有潜在作用，这一观点得到了诱变实验和分子动力学模拟的支持。这些发现加深了我们对环B在调节PL7藻酸盐裂解酶底物结合中关键作用的理解。
