Abstract
Lysozyme is widely used as a model protein in studies of structure–function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.
Highlights
Lysozyme has been widely used as a model protein to evaluate protein structure–function relationships [1], such as in studies of amyloid fibril disease [2] and as a preservative for foods and over-the-counter medicines [3]
We designed and evaluated a genetic enzyme screening system (GESS)-based high-throughput screening (HTS) method for bacteriophage T4 lysozyme (T4L) evolution, which can expand the applications of lysozyme from studies of protein structure–function relationships to
We designed and evaluated a GESS-based HTS method for bacteriophage T4L evolution, which can expand the applications of lysozyme from studies of protein structure–function relationships to biological degradation of secondary sludge
Summary
Lysozyme has been widely used as a model protein to evaluate protein structure–function relationships [1], such as in studies of amyloid fibril disease [2] and as a preservative for foods and over-the-counter medicines [3]. Biological hydrolysis of bacterial cells is a rate-limiting step in most anaerobic digestion processes for secondary sludge [8,9]. In conventional anaerobic digestion processes, multiple physicochemical approaches are used to enhance the hydrolysis efficiency of secondary sludge [10,11,12], including thermal pretreatment [13], ultrasonic technology [10], and acid/alkaline chemical pretreatment [14]. These physicochemical methods require large amounts of energy and corrosive equipment.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
