Directed Coevolution of Two Cellulosic Enzymes in Escherichia coli Based on Their Synergistic Reactions

Methods in Molecular Biology
Min LiuHongwei Yu

Abstract

Directed evolution is a widely used technique for improving enzymatic properties. The development of an efficient high-throughput screening method is a key procedure, which is however often unavailable for many enzyme reactions, including the cellulase-catalyzed cellulose hydrolysis. Here, we describe a high-throughput screening assay for directed coevolution of two cellulases (an endoglucanase and a β-glucosidase) in form of a bicistronic operon based on their synergistic reactions. Insoluble filter paper is used as the real cellulose substrate to screen for positive enzyme variants, facilitated by the colorimetric assay coupled to glucose liberated from cellulose under catalysis of endoglucanase and β-glucosidase. Directed coevolution saves the labor and time required for two independent directed evolution cycles, which might provide reference for the engineering of other cellulosic enzymes or multienzyme systems.

Citations

Dec 11, 2021·Applied Microbiology and Biotechnology·Ruiqin ZhangYanjun Li

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.