GENPLAT (GLBRC 효소 플랫폼)는 바이오 매스 열화에 대한 효소 칵테일의 발견과 최적화를위한 자동화 플랫폼입니다. 이것은 여러 feedstocks 및 여러 구성 요소를 포함하는 효소의 혼합물에 적용할 수 있습니다.
The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are “mixture” experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For “core” enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as “accessory” are set to 0%. “Core” and “accessory” are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).
그것은 널리 효소의 비용을 절감하는 경제적인 리그노셀룰로오스성 에탄올 산업의 발전에 중요하다는 인식됩니다. 현재 사용 가능한 상용 효소 칵테일은 여러 단백질 (Nagendran 외., 2009)의 복잡하고 제대로 정의 혼합물이며, 그들은 주로 산성 – pretreated 옥수수 여물에 사용에 대한 적응입니다. 더 효소 칵테일의 발전을 촉진하기 위해서는 여러 실험실 효소 발견과 특성화에 대한 높은 처리량 플랫폼을 개발했습니다. .; 데커 동부 표준시 로봇 효소 및 바이오 매스 slurries의 분배, 실험의 통계적 설계, 및 / 또는 Glc의 자동 결정 및 Xyl (베를린 외, 2007 :이 영역에서 노력도 GENPLAT에서 찾은 다음 속성 중 하나 이상을 포함해야 알 2009;. 김 외, 1998;. 킹 외, 2009).. GENPLAT는 대부분 6 구성 요소에서 분석할 수있는 효소 혼합물의 복잡 가장 크게, 이러한 이전의 노력을 확장최근 작품 이상의 16 이전 연구 (Banerjee 외., 2010c). 완만한은 엔드 오버 엔드 회전에 의해 소화하는 동안 믹싱, 및 GLU와 Xyl의 자동 colorimetric 결정 GENPLAT의 추가 주요 기능은 분배하는 동안 정지 여물의 slurries을 유지할 수 있습니다 비드 혼합 챔버 사용 (패들 저수지)입니다.
The authors have nothing to disclose.
이 작품은 에너지의 미국학과, 기본 에너지 과학 부문의 사무실에서 미국 에너지 대호 Bioenergy 연구 센터 계열 (과학 BER의 DOE 사무소 DE – FC02 – 07ER64494)과 부여 DE – FG02 – 91ER200021에 의해 부분적으로 투자되었다 화학 과학, Geosciences 및 Biosciences.의 우리는 그들의 자료와 개념적 공헌에 대한 존 스콧 – 크렉과 멜리사 Borrusch 감사드립니다.
Equipments and software used:
Reagents used: