Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome
Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome
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从绵羊瘤胃微生物组中分离的两种果胶酸裂合酶的克隆、表达和表征
DOI：
10.1007/s00253-022-12344-9
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发表时间：
2022-12
影响因子：
5
通讯作者：
Qian Wang
中科院分区：
文献类型：
--
作者：
Qian Deng;Shi-Qi Li;Xiao-Bao Sun;De-Ying Gao;Nuo Li;Hui-En Zhang;Zheng-Guang Wang;Jia-Kun Wang;Qian Wang
果胶裂解酶(Pectate Lyase，PEL)在降解植物初生细胞壁和中叶中起着重要作用，在工业上得到了广泛的应用。本研究从绵羊瘤胃微生物组中克隆了两个果胶裂解酶基因IDSPel16和IDSPel17。重组酶在大肠杆菌中表达，并对其功能进行了鉴定。IDSPel16和IDSPel17蛋白的最适温度为60℃，最适pH为10.0。IDSPel16在60℃以下相对稳定，在60℃预孵育1h后保持77.51%的残余活力，而IDSPel17在60℃下迅速变性，在pH 6.0~12.0范围内相对稳定，预处理1h后保持60%以上的残余活力。IDSPel16对多聚半乳糖醛酸有较高的活性，其Vmax为942.90 ±min68.11，而IDSPel17的Vmax仅为28.19Mol/ /mg， / /mg。反应产物分析表明，IDSPel17从底物中释放出不饱和的双半乳糖醛酸(UG2)和不饱和的三乳糖酸(UG3)，为典型的果胶内切酶(EC 4.2.2.2)。相反，IDSPel16最初生成不饱和寡聚半乳糖醛酸，然后将这些中间产物转化为uG2和不饱和半乳糖醛酸(UG1)作为最终产物，这是PEL中独特的解聚特征。据我们所知，IDSPel16发现了内切酶(EC 4.2.2.2)和外切酶(EC 4.2.2.9)的活性。这两种果胶裂解酶，特别是温度和pH相对稳定的IDSPel16，将在纺织、食品和饲料工业中具有潜在的应用价值。要点·从绵羊瘤胃微生物组中分离并鉴定了两个新的果胶裂解酶基因，IDSPel16和IDSPel17。·IDSPel16和IDSPel17都是碱性果胶酸裂解酶，可以从聚半乳糖醛酸中释放不饱和的双半乳糖醛酸和不饱和的三聚半乳糖醛酸酯。·IDSPel16，一种具有内切酶(EC 4.2.2.2)和外切酶(EC 4.2.2.9)活性的双功能果胶酸裂解酶，可能是一种潜在的工业应用候选。
Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes,IDSPel16andIDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed inEscherichia coliand functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ℃, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 °C, maintaining 77.51% residual activity after preincubation at 60 °C for 1 h, whereas IDSPel17 denatured rapidly at 60 °C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with aVmaxof 942.90 ± 68.11, whereas IDSPel17 had aVmaxof only 28.19 ± 2.23 μmol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG2) and unsaturated trigalacturonate (uG3) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2and unsaturated galacturonic acid (uG1) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo- and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries.Key points•Two novel pectate lyase genes, IDSPel16 and IDSPel17, were isolated and characterized from the sheep rumen microbiome.•Both IDSPel16 and IDSPel17 are alkaline pectate lyases, releasing unsaturated digalacturonate and unsaturated trigalacturonate from polygalacturonic acid.•IDSPel16, a bifunctional pectate lyase with endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities, could be a potential candidate for industrial application.
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影响因子：
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作者：
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