Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities

Abstract Objective To establish a temperature-induced chitosanase bacterial cell surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications. Results Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A and 2InaQ-N-CSN46A. The specific enzyme activity of 2InaQ-N-CSN46A reached 886.33±0.81 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. GPC revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 ℃. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs.Conclusions In this study, chitosanase was expressed on the surfaces of E. coli by increasing induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provided a novel strategy for industrial COSs production.

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Epitope Mapping of Monoclonal and Polyclonal Antibodies Using Bacterial Cell Surface Display

Methods in Molecular Biology - Monoclonal Antibodies ◽

10.1007/978-1-62703-992-5_29 ◽

2014 ◽

pp. 485-500 ◽

Cited By ~ 6

Author(s):

Anna-Luisa Volk ◽

Francis Jingxin Hu ◽

Johan Rockberg

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Epitope Mapping ◽

Polyclonal Antibodies ◽

Surface Display ◽

Cell Surface Display ◽

Bacterial Cell Surface

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Cell-Surface display of heterologous proteins: From high-throughput screening to environmental applications

Biotechnology and Bioengineering ◽

10.1002/bit.10407 ◽

2002 ◽

Vol 79 (5) ◽

pp. 496-503 ◽

Cited By ~ 70

Author(s):

Wilfred Chen ◽

George Georgiou

Keyword(s):

Cell Surface ◽

High Throughput ◽

High Throughput Screening ◽

Surface Display ◽

Cell Surface Display ◽

Environmental Applications ◽

Heterologous Proteins

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Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants

Applied and Environmental Microbiology ◽

10.1128/aem.66.2.788-793.2000 ◽

2000 ◽

Vol 66 (2) ◽

pp. 788-793 ◽

Cited By ~ 77

Author(s):

Yong-Sung Kim ◽

Heung-Chae Jung ◽

Jae-Gu Pan

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

High Throughput Screening ◽

Surface Display ◽

Bacterial Surface ◽

Bacterial Cell Surface ◽

Cell Extracts ◽

Cmcase Activity ◽

Bacterial Surface Display ◽

Variant Genes

ABSTRACT The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.

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