Transgalactosylation in a Water-Solvent Biphasic Reaction System with β-Galactosidase Displayed on the Surfaces of Bacillus subtilis Spores

Abstract

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that ␤-galactosidase (␤-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 ؋ 10 3 U/g (dry weight) of spores. The ␤-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed ␤-Gal, octyl-␤-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.

Production of N -Acetyl- d -Neuraminic Acid by Use of an Efficient Spore Surface Display System

“…The bacterial strains and plasmids used in this study are listed in Table 1 (20). Escherichia coli strains were grown in LuriaBertani (LB) medium at 37°C.…”

“…The use of isolated enzymes requires an extraction and a time-consuming purification or partial purification procedure. Although this problem can be overcome through the use of whole cells, the mass transfer limitation caused by the microbial cell membrane restricts the application of whole cells (20). Thus, a novel biocatalyst which excludes the permeability barrier and the timeconsuming enzyme purification procedure would represent a suitable alternative for Neu5Ac production.…”

“…Among the numerous systems that have been employed in surface display, bacterial spores offer considerable advantages because of their resistance to heat, radiation, and chemicals in a harsh environment (27). Enzymatic transgalactosylation by using the sporedisplayed enzyme as a catalyst has been reported (20).…”

CotC-CotU Heterodimerization during Assembly of the Bacillus subtilis Spore Coat

“…Recently, new functions have been assigned to the coat, from sensing the external environment through active enzymes present on its surface (4,12,24,40) to protecting the spore from predation by phagocytic protozoans (21). In addition, the coat is a novel system for the display at the spore surface of heterologous antigens (16,18,25), enzymes (22), and bioactive molecules (20).…”

Bacillus species are Superior Probiotic Feed-Additives for Poultry

“…28,33,34 This thermostability property allow incorporate the spores into the feed the during the milling and pelletizing of the feed and other applications as production of vaccines incorporate as adjuvants. 28,35,36 Spores also can be spray-dried. In addition, spores are resistant to desiccation and other stress factors like bile and low pHs both present in the gastric juice.…”