Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues

K Watanabe, K Kitamura, Y Suzuki
1996 Applied and Environmental Microbiology  
To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an
more » ... tely thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the ␤ turn and first turn of the ␣ helix) (. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases. Selection for E. coli colonies containing the B. coagulans oligo-1,6-glucosidase gene. Chromosomal DNA (3.0 g) of B. coagulans ATCC 7050 was partially digested with EcoRI and ligated into pUC18 (1.0 g). After transformation of E. coli C600 cells with the ligation mixture, about 2,000 ampicillin-resistant colonies were obtained. All E. coli cells with or without pUC18, pUC118, and pUC119 showed neither an isomaltose-hydrolyzing p-nitrophenyl-␣-D-glucopyranosidase active at 50ЊC nor a protein cross-reactive with rabbit antiserum against B. coagulans oligo-1,6-glucosidase on double immunodiffusion (30). A screening for E. coli C600 cells harboring the B. coagulans oligo-1,6-glucosidase gene was carried out by searching for p-nitrophenyl-␣-D-glucopyranosidase activity of transformant colonies on blotting filter paper (16). The method was performed according to the procedure described in our previous papers (30, 32), except that the incubation temperature of the paper disk was 50ЊC. As a result of the screening for oligo-1,6-glucosidase, one yellow colony, the color of which was caused by the reaction of oligo-1,6-glucosidase on the substrate, was selected. The screened clone carried a plasmid, pBCO1, containing a 4.3-kb DNA insert in pUC18. The physical map of the DNA insert, as revealed by restriction enzyme analysis, is depicted in Fig. 1 . Further subcloning resulted in two plasmids, pBCO2 and pBCO3, with inserts with sizes of 3.0 and 2.4 kb, respectively (Fig. 1) . Assay for the enzymatic activity of oligo-1,6-glucosidase. Oligo-1,6-glucosidase activity was determined spectrophotometrically at 50ЊC by monitoring the increase in A 400 of a reaction mixture (1.0 ml) containing 33.3 mM phosphate buffer (pH 6.8), 2 mM p-nitrophenyl-␣-D-glucopyranoside, and enzyme (25). One unit of activity was defined as the amount of enzyme hydrolyzing 1 mol of p-nitrophenyl-␣-D-glucopyranoside per min. Amino acid sequencing. An Applied Biosystems (Foster City, Calif.) 477A gas-liquid-phase protein sequencer was used for the determination of the aminoterminal sequence of the B. coagulans oligo-1,6-glucosidase. The purified sample (100 g) that reacted with phenylthiohydantoin was separated and identified with an Applied Biosystems on-line PTH 120A analyzer with a phenylthiohydantoin-C 18 high-performance liquid chromatography column. Purification of cloned oligo-1,6-glucosidase. All steps were carried out at 4ЊC, and centrifugation was done at 12,000 ϫ g unless otherwise stated. E. coli C600 cells bearing pBCO3 were cultivated overnight at 37ЊC in 5 ml of L broth supplemented with 50 g of ampicillin per ml. The culture (1 ml) was transferred into fresh medium (200 ml) and cultivated for 12 h. E. coli cells (wet weight, 14.2 g) obtained from the culture (total of 2 liters) by centrifugation for 10 min were suspended in 50 ml of buffer A (50 mM potassium phosphate, 5 mM EDTA [pH 7.0]) and disrupted by sonication at 4ЊC for 10 min. The cell debris was removed by centrifugation for 20 min. The sediments were then sonicated again. The cell extract (128 ml) was treated at 60ЊC for 30 min and centrifuged for 20 min. The supernatant was applied to a DEAE-cellulose column (5.0 by 20 cm) equilibrated with buffer A. Elution was performed at a rate of 20 ml/h with a linear gradient of 0 to 0.7 M NaCl in buffer A (2,000 ml). The active fractions were combined (380 ml), concentrated by ultrafiltration through a Diaflo-Amicon (Danvers, Mass.) PM-10 membrane and dialyzed against 150 mM phosphate-5 mM EDTA (pH 7.0 [buffer B]). The dialysate (20 ml) was loaded onto a Sephadex G-100 column (3.0 by 95 cm) equilibrated with buffer B. The column was developed with buffer B at the rate of 15 ml/h. The active fractions were combined (370 ml) and concentrated by ultrafiltration. The concentrate (74 ml) was dialyzed against 5 mM phosphate (pH 7.9) and applied to a hydroxylapatite (Bio-Gel HTP; Bio-Rad Laboratories, Richmond, Calif.) column (2.0 by 40 cm) equilibrated with the same buffer. After being washed with 120 ml of the same buffer, the column was eluted with 800 ml of a linear 5 to 50 mM phosphate buffer gradient. The active fractions were pooled (370 ml), concentrated by ultrafiltration, and 2072 WATANABE ET AL. APPL. ENVIRON. MICROBIOL. on May 4, 2020 by guest Downloaded from
doi:10.1128/aem.62.6.2066-2073.1996 fatcat:3vlmyiape5c7fj7whyivg3dnhq