Autolysis Resistance Of Thermophilic Protease WF146 And Its Application

Thermophilic protease WF146 is secreted by thermophile Bacillus sp. WF146 and belongs to the subtilisin family. The precursor of this enzyme contains the signal peptide, the N-terminal peptide, and the mature enzyme with a disulfide bond (Cys52-Cys65). After treatment at 60℃the pro-form enzyme can be self-processed into mature enzyme with a molecular mass of 32 kDa.The optimum temperature and pH of protease WF146 for catalysis are 85℃and pH 8.0, respectively. Protease WF146 is stable at 60℃in the presence of 10 mM CaCl2 and 10 mM NaCl, but suffers excessive autolysis in the presence of reducing agents or under high concentration (above 5μg/ml). This study focused mainly on revealing the autolysis mechanism of protease WF146 and improving its autolysis resistance. The autolysis-resistant mutant N63P/A66P was used in processing keratinous wastes and degrading prion proteins.First, the autolytic products were isolated from intact mature enzyme using size exclusive chromatography. By N-terminal sequencing two autolysis sites were identified at Asn63-Gly64 and Cys65-Ala66 which are located at a surface loop of the enzyme. The results of activity staining and activity assay showed that the autolytic products remained active. Furthermore, glutaraldehyde cross-linking and co-refolding experiments confirm that the autolytic products exist as complexes (FN1+FC1 or FN2+FC2). Although the specific activities of complexes at 60℃are similar to that of intact mature enzyme, their stabilities decreased substantially. That is the main reason why the stability of protease WF146 decrease when suffered autolysis. In order to delete the autolysis sites, we introduced the most rigid amino acid residue Pro at Asn63 and/or Ala66. As a result, the mutants N63P, A66P and N63P/A66P displayed improved stabilities in varying degrees. Among them, the double mutant N63P/A66P displayed a 2.8-fold or 2.2-fold longer half-life at 80℃under reduing condition or non-reduing condition, respectively, and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. These results indicate that the local flexibility of the surface loop is the main cause that leads to the autolysis, and strengthening of the local structure can reduce or even avoid the occurrence of autolysis.Keratin consists of tightly packed protein chains with high degrees of cross-linking of disulfide bonds, rendering it one of hard-to-degrade animal proteins. The results showed that, due to its improved stability, the mutant N63P/A66P was able to degrade feather at 80℃(-3 h) in the presence of reducing agents, with hydrolysis efficiency comparable to that of proteinase K at 50℃(-24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrPSc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes such as feather, and disinfection of medical apparatus.For the convenience of production and purification, Bacillus subtilis and Pichia pastor is were used as hosts for the expression of protease WF146. The recombinant enzyme was successfully expressed and secreted in these two hosts. The amount of recombinant enzyme secreted from B. subtilis and P. pastoris were 5μg/ml and 50μg/ml, respectively. In addition, the recombinant enzyme produced by P. pastoris was glycosylated, and showed an improved resistance to autolysis.