GENPLAT: Biyokütle Enzim Discovery ve Kokteyl Optimizasyonu Otomatik Platform
Summary
GENPLAT (GLBRC Enzim Platformu) biyokütle bozulması için enzim kokteyller keşif ve optimizasyonu için otomatik bir platform. Bu birden fazla hammadde ve birden fazla bileşen içeren enzimlerin karışımları adapte edilebilir.
Abstract
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).
Protocol
Discussion
Bu yaygın enzimlerin maliyetini azaltarak ekonomik bir lignocellulosic etanol endüstrisinin gelişimi için önemli olduğu kabul edilmektedir. Şu anda mevcut ticari enzim kokteyller birçok proteinleri (Nagendran ve ark, 2009) karmaşık ve kötü tanımlanmış karışımları ve özellikle asit-tedavi öncesi mısır koçanı kullanmak için uyarlanmıştır. Daha iyi enzim kokteyller gelişimini hızlandırmak için, bazı laboratuvarlar enzim keşif ve karakterizasyonu için yüksek verimli platformlar geliştirdik. Decker et robot, enzimler ve biyokütle çamurlar dağıtım, istatistiksel deney tasarımı ve / veya otomatik olarak belirlenmesi GLC ve Xyl (Berlin ve ark, 2007: Bu alandaki çabalar da GENPLAT bulunan aşağıdaki özelliklerden bir veya daha fazla dahil diğerleri, 2009; Kim ve ark, 1998; King ve ark, 2009). GENPLAT en az 6 bileşenleri analiz edilebilir enzim karışımlarının karmaşıklığı en önemlisi, bu daha önceki çabaları uzanıren son çalışma fazla 16 daha önceki çalışmalarda (Banerjee ve ark. 2010C). Üzerinde uç uç dönme sindirim sırasında nazik karıştırma; ve Glu ve Xyl otomatik kolorimetrik tespiti GENPLAT Ek temel özellikleri, dağıtım sırasında askıya koçanı çamurlar tutabilir boncuk karıştırma haznesinin kullanımı (kürek rezervuar).
Disclosures
The authors have nothing to disclose.
Acknowledgements
Bu çalışma, ABD Enerji Departmanı, Temel Enerji Bilimler, Bölüm Dairesi, ABD Enerji Great Lakes Biyoenerji Araştırma Merkezi Departmanı (DOE Bilim BER Office DE-FC02-07ER64494) ve hibe DE-FG02-91ER200021 tarafından finanse edildi Kimyasal Bilimler, Yer Bilimleri ve Biosciences. John Scott-Craig ve Melissa Borrusch Biz onların maddi ve kavramsal katkıları için teşekkür ederim.
Materials
Equipments and software used:
- Design–Expert software ,Version 8.0 (Stat-Ease, Inc., Minneapolis, MN) (http://www.statease.com)
- Biomek FXP laboratory automation workstation (Beckman-Coulter, Fullerton, CA)
- Biomek FXP software, Version 3.3 (Beckman-Coulter, Fullerton, CA)
- Paddle reservoir (Model VP 756C-1P100, V & P Scientific Inc., San Diego)
- Plates, 96-deep (1.5-ml) well (Abgene, Fisher Scientific, Epson, UK)
- Pierceable capmats (USA Scientific, Ocala, FL)
- Rotating hybridization incubator (Model 5420,VWR)
- Swinging-bucket centrifuge (Model 5810R, Eppendorf, Hamburg, Germany)
- Plates, Costar 96-well (Abgene, Fisher Scientific, Epson, UK)
- Plates, 384-well (BD Falcon, BD Biosciences, San Jose, CA)
Reagents used:
- Pichia pastoris and vectors: strain X33, and pPICZ and pPICZα (Invitrogen, Carlsbad, CA)
- T. reesei and vectors: strain QM9414 (US Department of Agriculture National Center for Agricultural Utilization Research, Peoria, IL), and pCB1004-EV (Banerjee et al., 2010b)
- Accellerase 1000 (lot number 1600844643) (Genencor, Inc., Rochester, NY)
- 50 mM sodium citrate buffer, pH 4.8
- Tetracycline stock: 10 mg/ml
- Cycloheximide stock: 10 mg/ml
- GOPOD assay kit , catalog K-GLUC (Megazyme, Bray, Ireland)
- Xylose assay kit, catalog K-XYLOSE (Megazyme, Bray, Ireland)
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