Abstract
BACKGROUND: Alicyclobacillus sp. A4 is thermoacidophilic and produces many glycoside hydrolases. An extremely acidic beta-1,4-glucanase (CelA4) has been isolated from Alicyclobacillus sp. A4 and purified. This glucanase with a molecular mass of 48.6 kDa decreases the viscosity of barley-soybean feed under simulated gastric conditions. Therefore, it has the potential to improve the nutrient bioavailability of pig feed. For the study reported herein, the full-length gene, CelA4, of this glucanase (CelA4) was identified using the sequences of six peptides and cloned from strain A4. The gene fragment (CelA4F) encoding the mature protein was expressed in Pichia pastoris. Sequence truncation and glycosylation were found for recombinant CelA4F, both of which affected the expression efficiency. The physical properties of various forms of CelA4 as they affected enzymatic activity were characterized. RESULTS: We located the full-length 2,148-bp gene for CelA4 (CelA4) in the genome of Alicyclobacillus sp. A4. CelA4 encodes a 715-residue polypeptide with a calculated molecular mass of 71.64 kDa, including an N-terminal signal peptide (residues 1-39), a catalytic domain (residues 39-497), and a C-terminal threonine-rich region (residues 498-715). Its deduced amino acid sequence and that of an Alicyclobacillus acidocaldarius endo-beta-1,4-glucanase were identical at 44% of the residue positions. When the experimental molecular mass of CelA4F--a recombinant protein designed to mimic the CelA4 sequence lacking the N-terminal signal peptide that had been expressed in Pichia pastoris--was compared with its hypothetical molecular mass, it was apparent that CelA4F was truncated, possibly at residue 497. An artificially truncated gene fragment (CelA4T) without C-terminal threonine-rich region was expressed in P. pastoris, and the expression efficiency of CelA4T was substantially greater than that of CelA4F. Purified CelA4F and CelA4T had similar molecular masses (~60 kDa) and enzymatic properties (optimum pH, 3.4; optimum temperature, 60 degrees C); they were relatively stable between pH 1.2 and 8.2 at 70 degrees C and resistant to acidic and neutral proteases. However, their molecular masses and thermostabilities differed from those of CelA4 isolated from Alicyclobacillus sp. A4. A deglycosylated form of CelA4 (CelA4D) had properties similar to that of CelA4 except that it was thermoliable at 60 degrees C. CONCLUSIONS: Truncation during expression of CelA4F or artificial truncation of its gene--both of which produced a form of CelA4 lacking a threonine-rich region that includes a putative linker--increased the level of enzyme produced in comparison with that produced by cultivation of Alicyclobacillus sp. A4. Glycosylation increased the thermostability of CelA4. Of the four forms of CelA4 studied, CelA4T was produced in highest yield and had the most favorable physical properties; therefore, it has potential for use in the feed industry.