In the field of enzyme engineering, the traditional plate screening method has always been a commonly used tool for researchers. However, this method has many limitations. Researchers need to cultivate and test enzymes one by one, which is like finding a needle in a vast sea, requiring a lot of time and effort,andefficiencycompareFor low. This leads to a longer screening time for traditional manual methods, and ultimately may not obtain a good mutant, making it difficult to meet the needs of practical applications.

Microfluidics of Tianmu BiotechnologyequipmentLike a "screening expert" with extraordinary abilities, capable of efficiently processing massive enzyme samples in a very short period of time, greatly improving screening efficiency. For example, if traditional methods can only process dozens of samples per day, microfluidic technology can process thousands of samples per day. At the same time, it is like a sharp eyed "observer" that can accurately capture the characteristic changes of each mutant, thus accurately screening out themHigher activity and stronger specificityThe enzyme. In addition,Tianmu Microfluidic PlatformIt has the characteristics of high integration, which can achieve automation and precise control of enzyme screening process, reduce the interference of human factors, and improve the reliability of screening results.

Summary of application directions of four published cases of microfluidic devices at present

Next,Select two typical cases from the numerous scientific research achievements published with the help of Tianmu Biological Microfluidic Equipment, and deeply explore their application and implementation in the field of enzyme engineering.

literature1

biosensor+Microfluidics:D-Breakthrough Improvement in the Activity and Stability of Aloulose Synthase

D-Aloketose is a low calorie rare ketohexose with physiological functions such as lowering blood sugar and antioxidation, and is a popular alternative sweetener in the food and pharmaceutical fields. Its biosynthesis depends on ketose3-Differential isomerase（KEases）However, existing enzymes have problems such as low catalytic efficiency and poor stabilityThis has restricted industrial production and application, and there is an urgent need to develop catalysts with higher catalytic efficiency and stronger stabilityKEasesTraditional screening methods, such as chromatographic analysis and microplate screening, have low efficiency and are difficult to meet the screening needs of high-throughput mutant libraries.

Professor Qin Huimin's team at Tianjin University of Science and Technology conducted rational design and directed evolution based on structural guidanceD-allulose3-Differential isomerase（SfDAE）Underwent renovation and constructed a system that includes approximately200A mutation library of ten thousand mutants and its useHigh throughput skin upgrade droplet single-cell sorting systemDREM cellcombineD-Genetic encoded biosensor for real-time monitoring of transcription regulatory factors responsive to alloketoseD-alluloseGenerate quantity, sort to obtain high-yield strains, expand cultivation, and perform enzyme activity detection.

DAEaseConstruction of mutant libraryandMicrofluidic screening process based on droplets and its application using high-performance liquid chromatographyDAEaseActivity Detection

after several roundsFADSThe SortingFinally, the mutant was identifiedM3-2Its catalytic efficiency is higher than that of the wild typeSfDAEimproved17Twice. mutantM3-2InD-Fructose isomerization toD-Aloulose exhibits higher catalytic efficiency and thermal stability in the reaction.

The time series diagram of the artificial mixed droplet library during the sorting process, displaying the detected fluorescence signals and fluorescence microscopy images of the droplets before sorting and the positive droplets after sorting

literature2

Bacterial biosensor+Microfluidics:Filter to get highactivitycolorAmino acid hydroxylase, Unlock5-HydroxytryptophanEfficient production

5- Hydroxytryptophan（5-HTP）It is an intermediate product of tryptophan metabolism and a precursor of the neurotransmitter serotonin, which has functions such as regulating emotions, improving sleep, and controlling appetite. Compared to plant extraction or chemical synthesis, biosynthesis5-HTPIt has the advantages of low cost and high efficiency. Natural tryptophan hydroxylase（TPH）There are problems of insufficient activity and poor stability in industrial applications, which limit their large-scale production.This study developed a method based onHigh throughput skin upgrade droplet single-cell sorting systemDREM cellMicrofluidic droplet sorting platform（DTSP）By combining bacterial biosensors, we have achievedTPHEfficient screening of mutants.

Professor Qin Huimin's team from Tianjin University has constructedTwo types of bacteria, a kind ofEnzyme producing bacteria（EPB）Used to expressTPHMutant,a kind ofDetecting bacteria（DB）Used for detectingL-Tryptophan（L-Trp）The concentration.Encapsulation of a single droplet within a liquid dropletEPBCells, proceedL-Trpto5-HTPAfter conversion, inject the detection bacteria into the droplet and determine the remaining amountL-TrpFluorescence signal sortingnHigh activity mutants were tested for their heat resistance and activity, and structural analysis was performed.

Ultra high throughput microfluidic screening platform based on droplets（DTSP）

useIterative saturation mutation（ISM）toTPHPerform directed evolution，Obtaining key mutations through multiple rounds of targeted mutation screeningbodyM4-1（D129L/Q132M/P103A/T236K），Catalytic activity enhancement4.25Doubling, improved thermal stability3.2timesDroplet microfluidic screening systemHourly selectable36Ten thousand mutants,The screening efficiency is much higher than traditional methods, and it has significant advantages in the screening process of large-scale mutation libraries.

Based on bacterial biosensors5-HPESchematic diagram of general process for high-throughput microfluidic droplet sorting(A)Encapsulation and cultivation of mutant libraries(B) DBCell injection and droplet culture(C)Positive droplet separation

These published practical cases allow us to more intuitively feel the enormous potential of microfluidic technology in the field of enzyme engineeringPotential and practical application value. These cases can not only provide valuable experience and reference for researchers, but also provide ideas and directions for the application of this technology in more scenarios.

Believe that with the development of Tianmu Biological MicrofluidicsequipmentThrough in-depth analysis of actual cases, we will further unlock their infinite possibilities in the field of enzyme engineering, promote enzyme engineering research to new heights, inject strong impetus into the development of the biotechnology industry, and enable more innovative achievements based on enzyme engineering to benefit human society. Let's look forward to discovering more surprising technological breakthroughs and application results in the subsequent case studies.