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从链霉菌 NL15-2K 中克隆和鉴定香草醛脱氢酶。

Molecular cloning and characterization of vanillin dehydrogenase from Streptomyces sp. NL15-2K.

机构信息

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima, 731-0153, Japan.

出版信息

BMC Microbiol. 2018 Oct 24;18(1):154. doi: 10.1186/s12866-018-1309-2.

DOI:10.1186/s12866-018-1309-2
PMID:30355315
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6201588/
Abstract

BACKGROUND

Streptomyces sp. NL15-2K, previously isolated from the forest soil, features an extensive catabolic network for lignin-derived aromatic compounds, including pathways transforming ferulic acid to vanillin, vanillic acid, and protocatechuic acid. To successfully use Streptomyces sp. NL15-2K as a biocatalyst for vanillin production, it is necessary to characterize the vanillin dehydrogenase (VDH) that degrades the produced vanillin to vanillic acid, as well as the gene encoding this enzyme. Here, we cloned the VDH-encoding gene (vdh) from strain NL15-2K and comprehensively characterized its gene product.

RESULTS

The vdh open reading frame contains 1488 bp and encodes a 496-amino-acid protein with a calculated molecular mass of 51,705 Da. Whereas the apparent native molecular mass of recombinant VDH was estimated to be 214 kDa by gel filtration analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a subunit molecular mass of ca. 56 kDa, indicating that VDH is a homotetramer. The recombinant enzyme showed optimal activity at 45 °C and pH 9.5. The VDH substrate specificity followed this order: vanillin (100%) > protocatechualdehyde (91%) > benzaldehyde (79%) > p-hydroxybenzaldehyde (56%) > isovanillin (49%) ≈ salicylaldehyde (48%) > anisaldehyde (15%) ≈ veratraldehyde (12%). Although peptide mass fingerprinting and BLAST searches indicated that this enzyme is a salicylaldehyde dehydrogenase (SALDH), the determined kinetic parameters clearly demonstrated that the enzyme is a vanillin dehydrogenase. Lastly, phylogenetic analysis revealed that VDH from Streptomyces sp. NL15-2K forms an independent branch in the phylogenetic tree and, hence, is evolutionarily distinct from other VDHs and SALDHs whose activities have been confirmed experimentally.

CONCLUSIONS

Our findings not only enhance the understanding of the enzymatic properties of VDH and the characteristics of its amino acid sequence, but also contribute to the development of Streptomyces sp. NL15-2K into a biocatalyst for the biotransformation of ferulic acid to vanillin.

摘要

背景

链霉菌 NL15-2K 是从森林土壤中分离出来的，其具有广泛的木质素衍生芳香族化合物的分解代谢网络，包括将阿魏酸转化为香草醛、香草酸和原儿茶酸的途径。为了成功地将链霉菌 NL15-2K 用作香草醛生产的生物催化剂，有必要对降解所产生的香草醛的香草醛脱氢酶 (VDH) 以及编码该酶的基因进行表征。在这里，我们从 NL15-2K 菌株中克隆了 VDH 编码基因 (vdh)，并全面表征了其基因产物。

结果

vdh 开放阅读框包含 1488bp，编码一个 496 个氨基酸的蛋白质，其计算分子量为 51705Da。尽管凝胶过滤分析估计重组 VDH 的表观天然分子量约为 214kDa，但十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示约 56kDa 的亚基分子量，表明 VDH 是一个四聚体。重组酶在 45°C 和 pH9.5 时表现出最佳活性。VDH 的底物特异性顺序为：香草醛 (100%)>原儿茶醛 (91%)>苯甲醛 (79%)>对羟基苯甲醛 (56%)>异香草醛 (49%)≈水杨醛 (48%)>茴香醛 (15%)≈藜芦醛 (12%)。尽管肽质量指纹图谱和 BLAST 搜索表明该酶是水杨醛脱氢酶 (SALDH)，但确定的动力学参数清楚地表明该酶是香草醛脱氢酶。最后，系统发育分析表明，链霉菌 NL15-2K 的 VDH 在系统发育树中形成一个独立的分支，因此在进化上与其他已证实具有活性的 VDH 和 SALDH 不同。

结论

我们的研究结果不仅增强了对 VDH 的酶学性质及其氨基酸序列特征的理解，还有助于将链霉菌 NL15-2K 开发为生物催化剂，用于将阿魏酸生物转化为香草醛。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/69270fdcf5da/12866_2018_1309_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/8e4997c669a2/12866_2018_1309_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/409b99db62da/12866_2018_1309_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/0bda8bb554bc/12866_2018_1309_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/5e1092ce4714/12866_2018_1309_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/e9d871d35426/12866_2018_1309_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/5f283757f5a2/12866_2018_1309_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/69270fdcf5da/12866_2018_1309_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/8e4997c669a2/12866_2018_1309_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/409b99db62da/12866_2018_1309_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/0bda8bb554bc/12866_2018_1309_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/5e1092ce4714/12866_2018_1309_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/e9d871d35426/12866_2018_1309_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/5f283757f5a2/12866_2018_1309_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37bf/6201588/69270fdcf5da/12866_2018_1309_Fig7_HTML.jpg

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