scholarly journals Widely applicable background depletion step enables transaminase evolution through solid-phase screening

2019 ◽  
Vol 10 (23) ◽  
pp. 5952-5958 ◽  
Author(s):  
Matteo Planchestainer ◽  
Eimear Hegarty ◽  
Christian M. Heckmann ◽  
Louise J. Gourlay ◽  
Francesca Paradisi
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Industrial Applications

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications.

scholarly journals Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

2017 ◽  
Author(s):  
Chenlu Zhang ◽  
Ligia Acosta-Sampson ◽  
Vivian Yaci Yu ◽  
Jamie H. D. Cate
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellulosic Biofuel ◽  
Economic Production ◽  
Report Analysis ◽  
Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

scholarly journals Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly

2018 ◽  
Vol 19 (10) ◽  
pp. 2989 ◽  
Author(s):  
Ji Zhang ◽  
Fuying Ma ◽  
Xiaoyu Zhang ◽  
Anli Geng
Keyword(s):  
Directed Evolution ◽  
Laccase Activity ◽  
Catalytic Properties ◽  
Random Mutagenesis ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Subunit Interaction ◽  
Cerrena Unicolor ◽  
Protein Model

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

scholarly journals Glassy Carbon: A Promising Material for Micro- and Nanomanufacturing

Materials ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 1857 ◽  
Author(s):  
Swati Sharma
Keyword(s):  
Glassy Carbon ◽  
Carbon Material ◽  
Large Scale ◽  
Solid Phase ◽  
Three Dimensional ◽  
Industrial Applications ◽  
Degradation Temperature ◽  
Pass Through ◽  
Semi Solid ◽  
Device Research

When certain polymers are heat-treated beyond their degradation temperature in the absence of oxygen, they pass through a semi-solid phase, followed by the loss of heteroatoms and the formation of a solid carbon material composed of a three-dimensional graphenic network, known as glassy (or glass-like) carbon. The thermochemical decomposition of polymers, or generally of any organic material, is defined as pyrolysis. Glassy carbon is used in various large-scale industrial applications and has proven its versatility in miniaturized devices. In this article, micro and nano-scale glassy carbon devices manufactured by (i) pyrolysis of specialized pre-patterned polymers and (ii) direct machining or etching of glassy carbon, with their respective applications, are reviewed. The prospects of the use of glassy carbon in the next-generation devices based on the material’s history and development, distinct features compared to other elemental carbon forms, and some large-scale processes that paved the way to the state-of-the-art, are evaluated. Selected support techniques such as the methods used for surface modification, and major characterization tools are briefly discussed. Barring historical aspects, this review mainly covers the advances in glassy carbon device research from the last five years (2013–2018). The goal is to provide a common platform to carbon material scientists, micro/nanomanufacturing experts, and microsystem engineers to stimulate glassy carbon device research.

scholarly journals A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

2001 ◽  
Vol 6 (4) ◽  
pp. 219-223 ◽  
Author(s):  
John M. Joern ◽  
Takeshi Sakamoto ◽  
Akira Arisawa ◽  
Frances H. Arnold
Keyword(s):  
Image Analysis ◽  
Directed Evolution ◽  
High Throughput ◽  
Digital Imaging ◽  
Solid Phase ◽  
High Throughput Screen ◽  
Microplate Reader ◽  
Dihydrodiol Dehydrogenase ◽  
Dioxygenase Activity

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-di- hydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30,uM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Combinatorial Mutagenesis Algorithms, Digital Imaging Spectroscopy, and Solid-Phase Assays for Directed Evolution

2003 ◽  
Author(s):  
Simon Delagrave ◽  
Edward Bylina ◽  
William Coleman ◽  
Steven Robles ◽  
Mary Yang ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Digital Imaging ◽  
Solid Phase ◽  
Imaging Spectroscopy ◽  
Combinatorial Mutagenesis

scholarly journals Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

2004 ◽  
Vol 70 (9) ◽  
pp. 5343-5348 ◽  
Author(s):  
John T. Lisle ◽  
Martin A. Hamilton ◽  
Alan R. Willse ◽  
Gordon A. McFeters
Keyword(s):  
Bacterial Abundance ◽  
Solid Phase ◽  
Fluorescent Microscopy ◽  
Industrial Applications ◽  
Bacterial Cells ◽  
Abundance Estimate ◽  
Statistical Confidence ◽  
Quality Of Water ◽  
Microscopic Count ◽  
Direct Counts

ABSTRACT Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter−1. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.

scholarly journals Characterization of Friction Surfaced Deposits on Low Carbon Steel

2019 ◽  
Vol 9 (1) ◽  
pp. 1248-1251
Keyword(s):  
Carbon Steel ◽  
Quality Evaluation ◽  
Solid Phase ◽  
Low Carbon Steel ◽  
Industrial Applications ◽  
Fusion Welding ◽  
Low Carbon ◽  
Friction Surfacing ◽  
Welding Processes

Surfacing With Friction Is A Process Derived From Friction Welding With Advantages Over Commercial Fusion Welding Processes, With Solid Phase Bonding. Here An Experiment Is Conducted To Produce Friction Surface Coating For Three Different Materials Like Aluminum And Stainless Steel And Tool Steel M2 Coating On Low Carbon Steel By Friction Surfacing. The Aim Of This Work Deposition Of Different Materials Is To Identify The Feasibility Of Friction Surfacing And Industrial Applications, Testing Of Deposits For Quality Evaluation Are Carried Out.

Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

ChemBioChem ◽  
2018 ◽  
Vol 19 (19) ◽  
pp. 2023-2032 ◽  
Author(s):  
Aitao Li ◽  
Zhoutong Sun ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Gene Synthesis ◽  
Mutant Library ◽  
Library Construction ◽  
The Future
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