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Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors
KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-5232-0805
KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
2013 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 9, 1754-1761 p.Article in journal (Refereed) Published
Abstract [en]

Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (K-m), the turnover number (k(cat)) and the enzyme inhibitor dissociation constants (k(i), k(i)'). Using this device we determine K-m and k(cat) for beta-galactosidase and the fluorogenic substrate Resorufin beta-D-galactopyranoside (RBG) to be 442 mu M and 1070 s(-1), respectively. Furthermore, we examine the inhibitory effects of isopropyl-beta-D-thiogalactopyranoside (IPTG) on beta-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

Place, publisher, year, edition, pages
2013. Vol. 13, no 9, 1754-1761 p.
Keyword [en]
Beta-Galactosidase, Microchip, Systems, Assays, Chip, Flow
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-121627DOI: 10.1039/c3lc41398eISI: 000316962700012Scopus ID: 2-s2.0-84875790638OAI: oai:DiVA.org:kth-121627DiVA: diva2:619346
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130503

Available from: 2013-05-03 Created: 2013-05-03 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Droplet microfluidics for directed evolution of biocatalysts
Open this publication in new window or tab >>Droplet microfluidics for directed evolution of biocatalysts
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysts, biologically derived catalysts, are of great importance for a wide range of industrial applications. They are used in the production of for example foods, pharmaceuticals and biofuels. Improving biocatalysts commonly relies on directed evolution, i.e. mutagenesis to form diverse variants followed by functional screening in an iterative fashion.

Droplet microfluidics is an emerging technology that can be applied for high throughput screening. A key feature of droplet microfluidics is the ability to encapsulate discrete objects, such as single cells, in picoliter-sized droplets at rates of over 1000 cells per second. Each droplet serves as a reaction vessel, analogous to a microwell, where a single clone can be screened.

In this thesis, droplet microfluidics is employed for directed evolution of biocatalysts. In paper I, a multiplexed droplet microfluidic method for characterization of enzyme variants is presented and validated by measuring the kinetics of β-galactosidase inhibited by IPTG. In paper II-III, a method for directed evolution of cells with improved production of industrially important enzymes is presented. Two rounds of directed evolution yielded improved strains. The strains had up to 6 times increased enzyme expression levels and whole-genome sequencing revealed 300 mutations, many of which mapped to the protein secretory pathway. In Paper IV, a method for directed evolution of enzyme variants under conditions lethal to host cells is developed. The method is used to screen for α-amylase variants with improved activity or stability at pH4. In Paper V, a method to screen cyanobacteria cell factories is developed and we show that the method can enrich for a strain with high production of L-lactate. In Paper VI, the metabolism of yeast cells encapsulated in microfluidic droplets is studied and found to depend on the choice of emulsion incubation device.

Taken together, droplet microfluidics is a promising technology for directed evolution of biocatalysts with the potential to vastly increase throughput and cut costs. The technology could pave the way for process customized biocatalysts and help replace polluting processes with sustainable green chemistry.

Abstract [sv]

Biokatalysatorer, dvs. katalysatorer av biologiskt ursprung, är viktiga inom en mängd olika industrier. För att förbättra biokatalysatorer används vanligtvis riktad evolution. En biokatalysator muteras för att skapa diversifierade varianter, vilka sedan kan screenas på funktion för att välja ut förbättrade varianter i ett iterativt arbetsflöde.

Droppmikrofluidik är en ny teknik som kan användas för höghastighetsscreening. Tekniken innebär att objekt såsom enskilda celler kan inkapslas i droppar med pikoliter volymer i hastigheter över 1000 celler per sekund. Varje droppe fungerar som ett reaktionskärl, analogt till en mikrobrunn, där en enskild klon kan screenas.

I denna avhandling redovisas studier om användning av droppmikrofluidik för riktad evolution av biokatalysatorer. I Paper I presenteras en multiplex droppmikrofluidisk metod för att karakterisera enzymvarianter. Metoden valideras genom att mäta kinetiken av β-galactosidase under inhibition av IPTG.  Paper II-III behandlar en metod för riktad evolution av celler med förbättrad produktion av industriellt viktiga enzymer. Två rundor av riktad evolution utfördes och förbättrade stammar kunde isoleras. Stammarna hade upp till 6 gånger högre enzymuttryck och hel genomsekvensering avslöjade fler än 300 mutationer. Många av dessa mutationer var i gener involverade i cellens maskineri för proteinsekretion. I Paper IV presenteras en metod för riktad evolution av enzymvarianter i miljöer som är dödliga för värdceller. Metoden användes för att screena efter förbättrade α-amylase varianter vid pH4. Paper V redovisar en metod för att screena cyanobakterier efter produktion av kemikalier. Vi visar att metoden kan användas för att anrika en stam som producerar mycket L-lactat. I Paper VI studeras metabolismen hos jästceller som är inkapslade i mikrofluidiska droppar. Cell metabolismen påverkades kraftigt av vilket inkubationskärl som användes för förvaring av emulsionen.

Sammanfattningsvis är droppmikrofluidik en lovande teknologi för riktad evolution av biokatalysatorer i och med att den ger avsevärt förbättrad screeninghastighet och lägre kostnader. Teknologin skulle kunna underlätta utvecklingen av ändamålsspecifika biokatalysatorer som potentiellt kan ersätta miljöförstörande processer med hållbar grön kemi.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 78 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:3
Keyword
Droplet microfluidics, Enzymes, directed evolution, high throughput screening
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-159286 (URN)978-91-7595-428-8 (ISBN)
Public defence
2015-02-20, Gard Aulan, Roslagstullsbacken, AlbaNova, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150129

Available from: 2015-01-29 Created: 2015-01-28 Last updated: 2015-01-29Bibliographically approved

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Jönsson, Håkan N.

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