Graciano Luciana, Corrêa Juliana Moço, Vieira Fabíola Giovanna Nesello, Bosetto Adilson, Loth Eduardo Alexandre, Kadowaki Marina Kimiko, Gandra Rinaldo Ferreira, Simão Rita de Cássia Garcia
Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil.
Appl Biochem Biotechnol. 2015 Apr;175(8):3915-29. doi: 10.1007/s12010-015-1560-z. Epub 2015 Mar 20.
Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for β-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, β-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 μM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.
新月柄杆菌（NA1000菌株）是水生细菌，能在营养质量低的环境中生存，拥有众多编码参与植物细胞壁解构的酶的基因，包括五个β-木糖苷酶基因（xynB1 - xynB5）和三个木聚糖酶基因（xynA1 - xynA3）。评估了在不同农业工业残渣存在下木聚糖酶的总体活性，发现玉米加工残渣在诱导细菌木聚糖酶方面最有效。xynA1基因（CCNA_02894）编码一种预测的糖水解酶第10组（GH10）木聚糖酶，将其克隆到载体pJet1.2 blunt中并亚克隆到表达载体pBAD/gIII后，使用0.02%阿拉伯糖在大肠杆菌LMG194中有效过表达，得到了一种含有羧基末端His标签的融合蛋白，命名为XynA1。纯XynA1的表征显示，以山毛榉木聚糖为底物时，其酶活性为18.26 U mL(-1)，比活性为2.22 U mg-(1)。XynA1的活性受到EDTA以及Cu(2+)和Mg(2+)等金属离子的抑制。相比之下，β-巯基乙醇、二硫苏糖醇（DTT）和Ca(2+)诱导重组酶活性。XynA1的动力学数据显示，K M和V max值分别为3.77 mg mL-(1)和10.20 μM min-(1)。最后，该酶的最适pH为6，最适温度为50℃。此外，XynA1在50℃孵育4小时后仍保持80%的活性，表明其对生物技术过程具有热稳定性。这项工作是关于新月柄杆菌木聚糖酶的克隆、过表达和酶学表征的首次研究。
