Protein stability subtilisin

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

Knowing that peptides and amines confer thermal stability on enzymes from certain thermophilic organisms (47-49) led some workers to examine protein stabilization by antibodies. It was found that the presence of specific polyclonal antibodies stabilize several enzymes (50, 51). In addition, not only did antibodies increjise the thermostability of a-amylase, glucoamylase, and subtilisin, but some stability toward acid denaturation, oxidizing agent, and organic solvent exposure was increased in specific cases (52, 53). 

The combination of molecular modeling with genetic engineering to enhance protein stability has been successful in certain cases. For instance, introducing carefully sited novel disulfide bonds increased protein stability in T4 lysozyme (11-13) and in X-repressor (14). However, the results in other proteins, for instance, in subtilisin (15,16) and in dihydrofolate reductase (17) have been less predictable. 

It is well known that the conjugation of proteins to chemical polymers can be used to improve protein stability or change its solubility. Ito et al. conjugated subtilisin to spiropyran carrying polymethacrylate... 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Transition-state stabilization in subtilisin is dissected by protein engineering... 

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

Tween 85 is used extensively for RME [84]. Russell and coworkers [234] used Tween 85/isopropanol microemulsions in hexane to solubilize proteins and not only showed >80% solubilization of cytochrome C at optimum conditions, but also proved that Tween 85 does not have a detrimental effect on the structure, function, and stability of subtilisin and cytochrome C. There are other reports available on the extraction and purification of proteins using Tween 85-RMs and also on the stability of protein activity in these systems [234]. It has also been shown that Tween 85-RMs can solubilize larger amounts of protein and water than AOT. Tween 85 has an HLB of 11, which indicates that it is soluble in organic solvents. In addition, it is biodegradable and can be successfully used as an additive in fertihzers [235,236]. Pfammatter et al. [35] have demonstrated that RMs made of Tween 85 and Span 80 can be successfully used for the solubilization and growth of whole cells. Recently, Hossain et al. [84] showed an enhanced enzymatic activity of Chromobacterium viscosum Hpase in AOT/Tween 85 mixed reverse micellar systems when compared to that in classical AOT-RMs. This is due to the modification of the interface in AOT-RMs caused by the co-adsorption of Tween 85, and increased availability of the oHve oil molecules (substrate) to the enzyme. 

MW 27,500) with no cofactors or metal ions reqnirement for its function, it displays Michaelis-Menten kinetics and it is secreted in large amounts by a wide variety of Bacillus species. Subtilisin is also among the most important industrial enzymes due to its use in laundry detergents. Protein engineering strategies for subtilisin have focused on a number of aspects, namely catalysis, substrate specificity, thermal and oxidative stability and pH profile. We will describe briefly each of these aspects. 

H. Bisgard-Frantzen, S. Carlsen, and J. M. Mikkelsen, Protein engineering of subtilisins to improve stability in detergent formulations, J. Biotechnol. 1993, 28, 55-68. 

In an Escherichia coli expression system for the aqualysin I precursor, the precursor is processed autoproteolytically into the mature 28-kDa enzyme by treatment at 65 ° C.23) In this case, the N-terminal pro-sequence is required for the production of active enzyme and functions to stabilize the precursor structure.283 The C-terminal pro-sequence is not essential for the production of active aqualysin 1,293 but seems to be involved in the translocation of the precursor across the cytoplasmic membrane.303 In a Thermus thermophilus expression system,313 the C-terminal pro-sequence is required for the production and extracellular secretion of active aqualysin I.323 In an E. coli expression system for the subtilisin E gene, the N-terminal pro-sequence is essential for the production of active enzyme,333 as in the case of aqualysin I. The requirement of the pro-sequence is also shown in vitro for the refolding of the inactive mature protein to an active enzyme.34 353 The functions of the N-terminal pro-sequences of aqualysin I and subtilisin E seem to be similar. 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Cunningham, B. C., and Wells, J. A. (1987). Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. Protein Eng, 1(4), 319-325. 

Bryan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliland GL, Howard AJ, Poulos TL (1986) Proteases of enhanced stability characterization of a thermostable variant of subtilisin. Proteins Structure, Function and Genetics. 1 326-334... 

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