bThe incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The halflife of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T m ) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K m and k cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications. Many microorganisms have successfully colonized extreme temperature environments ranging from Ϫ20°C to 122°C (1, 2). Enzymes from psychrophiles, mesophiles, and (hyper)thermophiles usually perform efficient catalysis at low, moderate, and high temperatures, respectively. Psychrophilic enzymes are characterized as cold active but heat labile, and these characteristics may arise from an increase in either the global or localized flexibility of enzyme structure (3, 4). Compared to their psychrophilic and mesophilic counterparts, (hyper)thermophilic enzymes generally exhibit enhanced conformational rigidity (5-7). However, some (hyper)thermophilic enzymes may combine local flexibility in their active site with high overall rigidity, thus making them more thermostable and cold active than their mesophilic counterparts (5-7).Accumulating evidence suggests that the cumulative effect of minor improvements of local interactions enhances the intrinsic stability of (hyper)thermophilic enzymes (5, 8). Identification of protein stabilization mechanisms is normally based on comparative studies of homologous enzymes that are adapted to different temperatures, on mutational analyses, on directed evolution and on computational methods (6, 9). The results of these studies have provided a rational basis for improving enzyme stability by sitedirected mutagenesis (SDM) (10). Nevertheless, the process of engineering enzymes for higher thermostability and activity remains important and difficult. One reason for this problem is that structural differences between homologous enzymes that are adapted to different tempe...
Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75-and 45-kDa mature forms involves both hetero-and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations.
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