Bacillus Cellulase Molecular Cloning, Expression, and Surface Display on the Outer Membrane of Escherichia coli

Abstract

One of the main challenges of using recombinant enzymes is that they are derived from genetically-modified microorganisms commonly located in the intracellular region. The use of these recombinant enzymes for commercial purposes requires the additional processes of cell disruption and purification, which may result in enzyme loss, denaturation, and increased total production cost. In this study, the cellulase gene of Bacillus licheniformis ATCC 14580 was cloned, over-expressed, and surface displayed in recombinant Escherichia coli using an ice-nucleation protein (INP). INP, an outer membrane-bound protein from Pseudomonas syringae, was utilized as an anchor linker, which was cloned with a foreign cellulase gene into the pET21a vector to develop a surface display system on the outer membrane of E. coli. The resulting strain successfully revealed cellulase on the host cell surface. The over-expressed INP-cellulase fusion protein was confirmed via staining assay for determining the extracellular cellulase and Western blotting method for the molecular weight (MW) of cellulase, which was estimated to be around 61.7 kDa. Cell fractionation and localization tests demonstrated that the INP-cellulase fusion protein was mostly present in the supernatant (47.5%) and outer membrane (19.4%), while the wild-type strain intracellularly retained enzymes within cytosol (>61%), indicating that the INP gene directed the cellulase expression on the bacteria cell surface. Further studies of the optimal enzyme activity were observed at 60 °C and pH 7.0, and at least 75% of maximal enzyme activity was preserved at 70 °C.

Keywords: Bacillus licheniformis; Pseudomonas syringae; cellulase; ice nucleation protein; surface anchoring; whole cell catalysis.

Figure 1

Targeted gene insertion and expression in E. coli. (A) The gel electrophoresis of amplified PCR cellulose products from B. licheniformis ATCC 14580 (lane 1) and INP from P. syringae KCTC 1832 (lane 2). M: 1 kb DNA marker; (B) SDS-PAGE analysis of the recombinant cells; M: standard protein size marker (molecular biomasses in kilodaltons), lane 1: the supernatant fraction of recombinant cell culture medium, lane 2: the total cell lysates of recombinant cell; (C) The purified fusion proteins following Ni-nitrilotriacetic acid (NTA)-sepharose resin treatment; M: standard protein size marker (kDa), lane 1: imidazole concentration of 20 mM in the binding buffer, lane 2: imidazole concentration of 50 mM in the binding buffer, lane 3: imidazole concentration of 100 mM in the binding buffer; (D) Western blot analysis of the purified fusion protein from SDS-PAGE results probed with anti-His-tag antibody, respectively.

Figure 2

Congo-red staining assay of wild-type B. licheniformis (A); non-cellulase producing E. coli (B); and recombinant E. coli harboring INP-cellulase genes (C); all of which were topped and cultivated on agar medium of 0.5% CMC at 30 °C for 18 h. The hollow clear zones around the colonies indicate the degradation of CMC as a result of the cellulolytic enzymes.

Figure 3

Relative cellulase activity analysis of various pH and temperature conditions. (A) Enzymatic hydrolysis of 0.5% CMC was carried out at 50 °C for 3 h with an agitation of 200 rpm; (B) Enzymatic hydrolysis of 0.5% CMC was carried out at pH 7.0 for 3 h with an agitation of 200 rpm. Error bars indicate the standard deviation of triplicated tests.