Secretory Expression of Rehmannia Class III Chitinase in Bacillus subtilis

Abstract

Chitinases (EC 3.2.1.14) are defined as enzymes cleaving a bond between the C1 and C4 of two consecutive N-actyl-D-glucosamines (GlcNAc) of chitin and have received much attention because of their wide range of applications such as protection of plants against the fungal pathogen [Sundheim et al., 1988] and particularly the production of chitooligosaccharides [Usui et al., 1990] due to their broad applications in medicine, agriculture, cosmetics, and food [Boller, 1986; Gooday, 1986; Ordentlich et al., 1988]. The JHIII gene (NCBI accession AY206666) encoding an acidic class III chitinase was isolated from the leaf of Rehmannia glutinosa (Korean name: ji-huang), which is commonly used in the oriental medicine [Kim and Pan, 2007]. To investigate the enzymatic properties of JHIII protein, the corresponding gene, JHIII, was first expressed in Escherichia coli. Although E. coli has been the most commonly employed in the production of heterologous proteins and many heterologous proteins have been expressed directly or as fusion proteins in the cytoplasm of E. coli, the recombinant JHIII (rJHIII) was in an extremely insoluble form that it showed little chitinolytic activity (data not shown). rJHIII was purified by immobilized metal affinity chromatography using a nickel agarose resin (His-bind, Merck, Germany) as a packing material under the denaturing conditions and used as an immunogen for the production of an antirJHIII antibody in the Balb b/c mouse. Other expression systems, such as the Bacillus expression system, were also explored to obtain the functional rJHIII. The greatest advantage of B. subtilis is its ability to secrete proteins directly into the culture medium. A further advantage of this direct secretion is that the proteins are often correctly folded, disulfide-bonded, and soluble. pUB140, a plasmid vector for the Bacillus expression, was used [Son et al., 2001]. However, in the present study, for the sake of convenient cloning, a shuttle vector was made between E. coli and B. subtilis. An origin of replication (ori) and a selection marker (amp) from pUC18 were introduced into pUB140, resulting in a shuttle vector pUBL140, which can be replicated in both B. subtilis and E. coli (Fig. 1A). For the purpose of overexpression and secretion of a foreign protein in B. subtilis, a promotor (amyR2) and a signal sequence (SS) were isolated from B. subtilis NA64, an α-amylasehyperproducing strain, [Yamazaki et al., 1983] and cloned into the p8A-1 plasmid. They were then inserted into pUBL140, resulting in pUBLamy (Fig. 1B). The promotor amyR2 is known to be transcribed at an early stationary phase of the cell growth. An expression vector, pUBLJHIII, was constructed to express JHIII gene in B. subtilis. The putative mature protein region of JHIII gene was amplified by PCR from the genomic DNA using the synthetic primers, JHB (5'-GGATCCAGGCAAAATCT CAATCTACTGG-3') and JHP (5'-CTGCAGTCAAACA TCCTTCTTGATAGAAG-3'). The PCR product was cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced. The JHIII gene fragment digested with BamHI and PstI was purified and inserted into the expression vector pUBLamy, which has been digested with the same restriction enzymes (Fig. 1B). The expression vector pUBLJHIII and the control vector pUBLamy were transformed into B. subtilis WB600 deficient in six extracellular proteases [Wu et al., 1991] according to the method described by Sadaie and Kada [1983] with a slight modification. To express the recombinant enzymes, B. subtilis was inoculated into 50 mL LB media containing 10 μg/mL kanamycin and grown at 37oC for 16 h with shaking at 300 rpm. All overnight cultures were inoculated into the jar fermentor (MK250, Korea Fermentor Co., Incheon, Korea) containing 2 L LB media without antibiotics. The fermentor operating conditions were: 37oC with shaking at 500 rpm, and 3 kgf/cm2. The cell growth and the *Corresponding author Phone: +82-33-650-7115; Fax: +82-33-650-7299 E-mail: panc@kist.re.kr