Automated saccharification assay for determination of digestibility in plant materials.
Automated saccharification assay for determination of digestibility in plant materials.
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DOI：
10.1186/1754-6834-3-23
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发表时间：
2010-10-27
影响因子：
6.3
通讯作者：
McQueen-Mason SJ
中科院分区：
文献类型：
--
作者：
Gomez LD;Whitehead C;Barakate A;Halpin C;McQueen-Mason SJ
细胞壁阻力是生产第二代生物燃料的主要障碍。木质纤维素的分解可以通过发酵为生产燃料或其他工业产品提供糖。了解细胞壁抵抗消化的生化基础将有助于开发更有效和更经济的方法从生物质中生产糖。一种方法是利用关联遗传学鉴定在生物量抗性中起作用的植物基因。这种方法需要一种强大而可靠的高通量（HT）测定生物质消化率的方法，该方法可用于筛选此类研究中涉及的大量样品。我们开发了一种基于机器人平台的高温糖化实验，可以在96孔板格式中进行酶解和释放糖的定量。生物质粉末的称重和成型由一个机器人工作站完成，在那里，植物材料被磨碎，运送到板中的所需孔中，并以0.1毫克的精度称重。一旦板装载，自动液体处理平台提供可选的轻度预处理（< 100°C），然后是生物质的酶解。然后分析水解产生的等分物以释放还原糖当量。同一平台可用于不同酶和酶鸡尾酒的比较评价。通过测量木质素修饰烟草茎秆的糖化过程，对平台的灵敏度和可靠性进行了评价，并对自动分析和人工分析结果进行了比较。自动化分析系统灵敏、稳健、可靠。该系统可以可靠地检测植物组织糖化的差异，并且能够在最少的人为干预下处理大量样品。自动化系统发现某些木质素修饰系的消化率显著增加，其方式与木质素修饰对细胞壁特性的已知影响相兼容。我们得出的结论是，这种自动化分析平台具有足够的灵敏度和可靠性，可以进行突变鉴定和遗传关联研究所需的大量植物群体的筛选。
Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.
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影响因子：
5.4
作者：
Hall, Melanie;Bansal, Prabuddha;Bommarius, Andreas S.
通讯作者：
Bommarius, Andreas S.
影响因子：
3.8
作者：
Studer, Michael H.;DeMartini, Jaclyn D.;Wyman, Charles E.
通讯作者：
Wyman, Charles E.
DOI：
10.1073/pnas.0907549107
发表时间：
2010-01-12
影响因子：
11.1
作者：
Lionetti, Vincenzo;Francocci, Fedra;Cervone, Felice
通讯作者：
Cervone, Felice
影响因子：
2.9
作者：
HONDA, S;NISHIMURA, Y;KAKEHI, K
通讯作者：
KAKEHI, K
影响因子：
6.3
作者：
Gomez LD;Bristow JK;Statham ER;McQueen-Mason SJ
通讯作者：
McQueen-Mason SJ