Chitinase Genes LbCHI31 and LbCHI32 from Limonium bicolor Were Successfully Expressed in Escherichia coli and Exhibit Recombinant Chitinase Activities

Abstract

The two chitinase genes, LbCHI31 and LbCHI32 from Limonium bicolor, were, respectively, expressed in Escherichia coli BL21 strain. The intracellular recombinant chitinases, inrCHI31 and inrCHI32, and the extracellular exrCHI31 and exrCHI32 could be produced into E. coli. The exrCHI31 and exrCHI32 can be secreted into extracellular medium. The optimal reaction condition for inrCHI31 was 5 mmol/L of Mn2+ at 40°C and pH 5.0 with an activity of 0.772 U using Alternaria alternata cell wall as substrate. The optimal condition of inrCHI32 was 5 mmol/L of Ba2+ at 45°C and pH 5.0 with an activity of 0.792 U using Valsa sordida cell wall as substrate. The optimal reaction condition of exrCHI31 was 5 mmol/L of Zn2+ at 40°C and pH 5.0, and the activity was 0.921 U using the A. alternata cell wall as substrate. Simultaneously, the optimal condition of exrCHI32 was 5 mmol/L of K+ at 45°C and pH 5.0, with V. sordida cell wall as the substrate, and the activity was 0.897 U. Furthermore, the activities of extracellular recombinant enzymes on fungal cell walls and compounds were generally higher than those of the intracellular recombinant enzymes. Recombinant exrCHI31 and exrCHI32 have better hydrolytic ability on cell walls of different fungi than synthetic chitins and obviously showed activity against A. alternata.

Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N‐acetyl glucosamine and in biocontrol of phytopathogenic fungi

“…Upon treatment with recombinant chitinase, perforations in mycelium and deformed spores were observed in F. oxysporum and C. lunata , when examined under electron microscopy (Figure ). The release of chitooligosaccharides from the fungal cell wall and leakage of cytoplasmic contents are responsible for the inhibition of the growth of phytopathogenic fungi, as reported with chitinases of B. cereus YQ 308 and Limonium bicolor in the biocontrol of F. oxysporium .…”

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

“…Like Liu et al expressed two chitinase genes, LbCHI31 and LbCHI32 from Limonium bicolor in E. coli BL21 strain and it has significant inhibition against A. alternate . 39 In a similar study, Itoh et al overexpressed a cell surface-expressed chitinase from Paenibacillus in E. coli . 40 In another study, horsetail derived chitinase gene was overproduced in E. coli .…”

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

“…In contrast, empty vector control cells grew vigorously, as did cells expressing ChiA for 2–6 h, confirming that the main bactericidal factor was the long-term expression of ChiA. E. coli is already used for the production of recombinant chitinase and the product accumulates in the cytoplasm, but even in these cases some chitinase was detected in the extracellular environment, suggesting a certain degree of cell lysis [ 47 , 48 , 49 ]. These publications focused on overall expression levels or screening in agar plate or MTP assays, where a population of >10 8 identical clones was assayed per well or spot, therefore cell lysis was not a concern.…”