Department of Energy, Great Lakes Bioenergy Research Center, Michigan State University, E. Lansing, Michigan 48824, USA.
Biotechnol Bioeng. 2010 Aug 1;106(5):707-20. doi: 10.1002/bit.22741.
The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of "accessory" proteins for development of superior enzyme mixtures for biomass conversion.
酶的高成本是阻止经济可行的木质纤维素乙醇产业发展的主要瓶颈。用于将植物生物质转化为可发酵糖的商业酶混合物是复杂的混合物，其中含有 80 多种活性和相对比例不佳的蛋白质。作为开发更有效的生物质转化用酶混合物的一步，我们开发了一个平台，称为 GENPLAT，该平台使用机器人液体处理和统计学有效的实验设计来分析合成酶混合物。使用商业酶（Accellerase 1000 +/- Multifect Xylanase 和 Spezyme CP +/- Novozyme 188）对系统进行测试，并作为比较基准。使用氨纤维膨胀（AFEX）预处理的玉米秸秆，粉碎至 0.5 毫米，葡聚糖负载为 0.2%，酶负载为 15mg 蛋白/g 葡聚糖，在 50°C 下消化 48 小时，商业酶分别释放出 53%和 41%的可用葡萄糖和木糖。系统优化了三种、五种和六种纯 Trichoderma 种的酶的混合物，在毕赤酵母中表达。为优化葡萄糖、木糖和 24 和 48 小时两个消化时间的葡萄糖+木糖的平均值，建立了统计模型。所得模型具有统计学意义（P < 0.0001），并表明葡萄糖释放的最佳组成（优化木糖释放的值在括号中）为 29%（5%）纤维二糖水解酶 1、5%（14%）纤维二糖水解酶 2、25%（25%）内切-β1,4-葡聚糖酶 1、14%（5%）β-葡萄糖苷酶、22%（34%）内切-β1,4-木聚糖酶 3 和 5%（17%）β-木糖苷酶，在 48 小时内，在 15mg/g 葡聚糖的蛋白负载下。比较两种经 AFEX 处理的玉米秸秆，粉碎至不同粒径，结果表明粒径（100 与 500 微米）对总消化率有很大影响。该测定平台和优化的“核心”集一起为快速测试和优化来自其他微生物和重组来源的替代核心酶以及测试用于生物质转化的优越酶混合物的“辅助”蛋白提供了一个起点。
