描述一个简单,快速的方法确定了大量的植物生物量的样品糖化潜力。这种分析的自动化平台,涉及植物生物量在96孔板和随后的表现预处理,水解和释放糖的定量分析的准备。
多糖,使植物木质纤维素生物质可以分解,产生的范围,随后可用于建立生物炼制的糖。这些原料将构成一个新的产业平台,这是可持续发展和碳中性,以取代目前对化石燃料的的依赖。在木质纤维素材料中观察到的解构顽抗是由植物细胞壁的内在属性。结晶纤维素嵌入xylans和arabinoxylans矩阵如多糖,整个结构是由木质素酚醛树脂聚合物,这也是难以消化1包裹。为了提高我们需要发现为细胞壁的糖化主要瓶颈,也屏幕突变和繁殖种群,以评估糖化2变异的植物原料的消化率。这些任务需要高吞吐量的方法,在这里,我们提出了一个分析平台,可以在96孔板格式执行糖化分析。这个平台已经发展到允许筛选不同植物物种的大量人口的纤维素消化率。我们温柔的预处理,部分酶解和糖的决心反应体积缩小,使大批自动化系统迅速进行评估。
这种自动化平台毫克的生物量,在受控条件下进行球磨,以减少植物材料,在一个可重复的方式进行了标准化的粒径。一旦样品的地面,自动格式化机器人分配到相应的水井深96孔板(图1)指定的材料和记录的金额。通常情况下,我们免除进入4口井有4分析重复相同的材料。一旦板块与植物所需的布局材料填充,他们是手动移动到液体处理站(图2)。在此站的样品受到与任稀酸或碱性温和的预处理,在孵育温度高达90 ° C随后被删除的预处理解决方案,并退回给适宜的pH为水解的样品用缓冲液冲洗。样本,然后培养的时间变长酶的混合物在50 ° C。一个等分是从水解还原糖自动MBTH比色法确定。
Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.
The authors have nothing to disclose.
作者想感谢一个Viksø尼尔森(诺维信公司)的纤维素酶的礼物。这项工作是由欧盟第七框架计划重建和BB/G016178和BB/G016194 BBSRC项目资助。
Name of the reagent | Company | Catalogue number |
---|---|---|
Grinding & weighing robot | Labman Automation | |
2 ml micro tube with caps | Sarstedt Ltd | 72.694 |
5 mm stainless steel beads | Qiagen Ltd | 69989 |
1.2ml Abgene square well storage plates | Fisher Scientific Ltd | TUL-050-050C |
Whatman cap mat for 96 square well plates | Fisher Scientific Ltd | 7704-0104 |
Liquid hanling robot | Tecan Group Ltd. | Freedom Evo 200 |
Sulphuric acid | Fisher Scientific Ltd | S/9231/PB17 |
Sodium hydroxide | Fisher Scientific Ltd | BPE359-500 |
Sodium acetate | Sigma-Aldrich | S8750-500G |
Acetic acid | Fisher Scientific Ltd | A/0420/PB17 |
Novozyme 188 | Novozyme | DCN00214 |
Celluclast 1.5L | Novozyme | CCN03122 |
96 well PCR full skirt plates | Sarstedt Ltd | 72.1980.202 |
D-Glucose | Fisher Scientific Ltd | G/0450/53 |
3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate | Sigma-Aldrich | 129739-25G |
DL-dithiothreitol | Sigma-Aldrich | D9163-1G |
Corning microplate 96 well flat bottom | Fisher Scientific Ltd | TKT-521-050H |
Ammonium iron (III) sulfate dodecahydrate | Sigma-Aldrich | F1668-250G |
Sulfamic acid | Sigma-Aldrich | 242772-500G |
Hydrochloric acid | Fisher Scientific Ltd | 12462-0026 |