फफूंद Endoglucanase में गतिविधि का उच्च Throughput स्क्रीनिंग Escherichia कोलाई</em
Summary
हम कवक endoglucanase में गतिविधि के लिए स्क्रीन के लिए एक कम लागत, उच्च throughput के विधि का वर्णन<em> ई. कोलाई.</em> विधि सब्सट्रेट गिरावट का एक साधारण दृश्य readout पर निर्भर करता है, एंजाइम शुद्धि की आवश्यकता नहीं है, और उच्च स्केलेबल है. यह एंजाइम वेरिएंट के बड़े पुस्तकालयों के तेजी से स्क्रीनिंग के लिए अनुमति देता है.
Abstract
Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12.
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (“halos”) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness.
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.
Protocol
Discussion
प्रोटोकॉल यहाँ वर्णित न्यूनतम हेरफेर के साथ तेजी से और उच्च सक्रिय एंजाइमों के throughput पहचान सक्षम बनाता है. गतिविधि का पता लगाने काफी संवेदनशील है, और गुणात्मक कक्ष के भीतर गतिविधि की राशि को दर्शाता है. ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम गॉर्डन और बेट्टी मूर फाउंडेशन, द्वारा और / UNCF मर्क विज्ञान पहल द्वारा वित्त पोषित किया गया था.
Materials
| Reagent | Company | Product number |
|---|---|---|
| IPTG | Sigma | I1284 |
| Congo Red | Sigma | C6277 |
| Carboxymethyl Cellulose | Sigma | 360384 |
| NaCl | Sigma | S1679 |
| Bacto-agar | BD Biosciences | 214030 |
| Bioassay plates | Thermo Scientific | 240845 |
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