Griseus Trypsin Research Articles

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution-guided mutagenesis

Streptomyces griseus trypsin (SGT) is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions. However, SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue. In this study, by engineering the α signal peptide in Pichia pastoris, we increased the SGT amidase activity to 67.91 U mL−1 in shake flask cultures. Afterwards, we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S, V177I and E180M with increased catalytic efficiencies. On this basis, we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL−1 in shake flasks and 2506 U mL−1 in 3-L fed-batch cultures. Moreover, single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5, which was 2 times better than the animal-sourced trypsin.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor

BackgroundTrypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate.ResultsThe tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL−1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg−1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL−1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris.ConclusionsSGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris

Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the α-factor signal peptide, trypsin production (amidase activity) was improved to 177.85±2.83U·mL−1 in a 3-L fermenter (a 3.75-fold increase). Subsequently, all of the residues involved in autolysis that were identified by mass spectrometry were mutated and the resulting proteins were evaluated. In particular, the variant tbcf (K101A) demonstrated high stability and production (227.65±6.51U·mL−1 and 185.71±5.68mg·L−1, respectively). The recombinant strain constructed here has great potential for large-scale production of active trypsin.

Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy.

N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml−1 (amidase activity, 8532 ± 142.2 U·ml−1 BAEE activity) with a productivity of 0.49 U·ml−1·h−1, which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.

Improvement of catalytic efficiency and thermostability of recombinant Streptomyces griseus trypsin by introducing artificial peptide

Streptomyces trypsin is one of the serine proteinases in Streptomyces griseus and acts as a key mediator during cell growth and differentiation. S. griseus trypsin (SGT) could be successfully expressed in Pichia pastoris by engineering the natural propeptide APNP. In this study, the recombinant Exmt with peptide YVEF and the wild-type SGT were comparatively investigated in detail. The recombinant Exmt showed significantly increased thermostability which t(½) value was 3.89-fold of that of the SGT at 40 °C. Moreover, the catalytic efficiency (referring to the specificity constant, k cat/K m) and pH tolerance of Exmt were also improved. In silico modeling analysis uncovered that introduction of the peptide YVEF resulted in a broadened substrate binding pocket and closer catalytic triad (His⁵⁷, Asp¹⁰² and Ser¹⁹⁵). The intramolecular Hydrogen bonds and the cation π-interactions were also dramatically increased. The results indicated that engineering of the N-terminus with artificial peptides might be an effective approach for optimizing the properties of the target enzymes.

The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates

Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites, but topological specificities (i.e. recognition at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin (SGT) for high molecular mass substrates differ greatly, even though the enzymes have similar primary structures. In this study, we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants, we found that residue 71 of SOT, which is separate from the catalytic triad, contributes to the topological specificity. Using site-directed mutagenesis, residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation.

Medium Optimization and Application of Affinity Column Chromatography for Trypsin Production from Recombinant Streptomyces griseus

The expression vector pWHM3-TR1R2, which contains sprT encoding Streptomyces griseus trypsin (SGT) and two positive regulatory genes (sgtR1 and sgtR2), was introduced into S. griseus IFO13350 and the productivity of SGT by the transformant was investigated in various media. Among the tested media, Ferm-0 gave 1.4 times more trypsin activity than C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 medium with 2% dextrin and 1% tryptone (named as Ferm-II medium) yielded significantly enhanced trypsin activity, by 4.1-fold, than that of Ferm-0. For simplifying the purification process, the cultural supernatant of S. griseus transformant in Ferm-II medium was fractionated with ammonium sulfate (25%-55%), and then applied to Hitrap benzamidine FF affinity column chromatography. The specific activity of the purified SGT by one-step column chromatography was 69,550 unit/mg protein, and the overall purification yield was above 8%, which was more effective than the methods of previous reports. The trypsin activity of the purified SGT was most active at pH 8.0 and 50 degrees C, and maintained their activities between pH 7.0 and pH 9.0, and up to 70 degrees C. These enzymatic properties were very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

Two bacterial collagenolytic serine proteases have different topological specificities

From among 2000 soil isolates, we purified a secreted serine protease from Streptomyces omiyaensis (SOT), which has extremely high gelatinolytic activity. Using sequence analysis, the primary structure of SOT showed 77% identity with that of S. griseus trypsin (SGT). We constructed recombinants SOT and SGT using S. lividans. They indicated similar properties on optimum pH and temperature, thermostability, and substrate preference using fluorescence energy transfer combinatorial libraries. SOT greatly hydrolyzed both type I and type IV collagens, but SGT has poor ability to hydrolyze type IV collagen. Furthermore, SOT exhibits higher hydrolytic activities toward other protein substrate such as gelatin and casein than SGT. These results suggest that these two enzymes have different topological specificities in spite of their similar primary structures. We also constructed chimeras between SOT and SGT to investigate which domain is associated with differences in their substrate specificity. In comparison to substrate specificities of chimeras, we found that the N-terminal domain contributes to the determination of topological specificity.

Trypsin-Catalyzed Synthesis of Dipeptide Containing .ALPHA.-Aminoisobutylic Acid Using p- and m-(Amidinomethyl)phenyl Esters as Acyl Donor

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An alpha-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was studied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Streptomyces griseus trypsin is stabilized against autolysis by the cooperation of a salt bridge and cation-pi interaction.

Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lacks the conserved disulphide bond surrounding the autolysis loop. We investigated the molecular mechanism by which SGT is stabilized against autolysis. The autolysis loop connects to another surface loop via a salt bridge (Glu146-Arg222), and the Arg222 residue also forms a cation-pi interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 is the main force stabilizing the enzyme against autolysis. The effect of the cation-pi interaction at Tyr217-Arg222 is small, however, its presence increases the half-life by about five hours and enhances the protein stability more than three-fold considering the catalytic activity in the presence of the salt bridge. The melting temperature also showed cooperation between the salt bridge and cation-pi interaction. These findings show that S. griseus trypsin is stabilized against autolysis through a cooperative network of a salt bridge and cation-pi interaction, which compensate for the absence of the conserved C136-C201 disulphide bond.

The first semi-synthetic serine protease made by native chemical ligation

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure–function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.

Immobilization of Protein Involving Disulfide Bond for Quantitative Evaluation of Refolding Media.

In order to select adequate solvents for protein refolding, immobilized preparation of a given protein that is covalently fixed on the surface of support materials, has been employed. This method is greatly useful for selection of solvents appropriate to refolding itself of the protein molecule, excluding unfavorable intermolecular interactions such as aggregation and autoproteolysis that emerge concurrently during the refolding. The purpose of this work is to obtain the preparation that warrants the constant recovered activity in the same denaturation/renaturation procedure. Such immobilized preparation is necessary for quantitative estimation of ability of refolding solvents. The globular proteins employed were subtilisin BPN’, ribonuclease A and Streptomyces griseus trypsin. In the case of such a protein as subtilisin possessing no disulfide bond in the molecule, agarose beads which have been widely used exhibited high retained activity and the constant recovered activity in the repeated denaturation/renaturation procedures, and revealed to be excellent as a support material. However, agarose gels were not suitable in the case of the other enzymes possessing disulfide bonds. This is probably attributable to involvement of reduction and oxidation reactions in the procedure applied to the latter case. Investigation of various support materials other than agarose beads revealed that the stable immobilized preparation of disulfide-involving protein could be established by use of glass beads that are resistant against oxidation and reduction with relatively high retained activity.

Trypsin-catalyzed peptide synthesis with m-guanidinophenyl and m-(guanidinomethyl)phenyl esters as acyl donor component.

Two series of inverse substrates, m-guanidinophenyl and m-(guanidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)-amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.

High performance in refolding of Streptomyces griseus trypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor.

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 microg/ml of SGT did not exceed 15%. This low yield was assumed to be due mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refolding media were examined for their ability to minimize misfolding. As a result, 1 M Tris, 1 M diethanolamine, and 1 M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding. The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.

Enzymatic peptide synthesis with p-guanidinophenyl and p-(guanidinomethyl)phenyl esters as acyl donors.

Two series of "inverse substrates", N-Boc-amino acid p-guanidinophenyl and p-(guanidinomethyl)phenyl esters, were prepared as acyl donor components for enzymatic peptide synthesis. The kinetic behavior of these esters toward bovine and Streptomyces griseus (SG) trypsin was analyzed. The spatial requirement of the active site of these enzymes for catalytic efficiency is discussed based on the steric characteristics of the substrates. These substrates were found to couple readily with amino acid p-nitroanilides to produce peptides. SG trypsin was the most efficient catalyst among the enzymes tested (bovine, porcine, and SG trypsin).

Enzymatic coupling of α,α-dialkyl amino acids using inverse substrates as acyl donors

Two series of inverse substrates, N α -Boc- α, α-dialkyl amino acid p-guanidino- and p-(guanidinomethyl)phenyl esters, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with amino acid p-nitroanilide to produce peptide. Streptomyces griseus trypsin was a more efficient catalyst than bovine trypsin.

Crystal structure of Streptomyces erythraeus trypsin at 1.9 A resolution.

A trypsin-like serine protease from Streptomyces erythraeus (abbreviated as SET) has been crystallized at pH 7, which is within its active pH range. The crystal structure of SET has been solved by molecular replacement using the atomic model of Streptomyces griseus trypsin (SGT), which is 37% homologous with SET, and refined to the crystallographic R factor of 0.199 for 15,878 reflections with Fo/sigma(F) > 3 between 7 and 1.9 A resolution. The final model of SET contains 1,619 protein atoms and 97 water molecules. No Ca2+ ion is present in SET apparently because (i) the two carboxylate groups from two Glu residues, which bind a Ca2+ ion in bovine trypsin (BT) or SGT, have disappeared; and (ii) a guanidino group from an Arg residue is unfavorably present in the potential binding region. There is an unusual type II beta-turn in which the third residue is Asp instead of Gly. This Asp residue is the only non-Gly residue significantly outside the allowed regions in the Ramachandran map. The three-dimensional structure of SET is essentially the same as those of other trypsins of mammalian origin. The 211 C alpha atoms of SET exhibit an r.m.s. deviation of 1.16 A with equivalent atoms of BT, and the 208 equivalent C alpha atoms between SET and SGT exhibit an r.m.s. deviation of 1.09 A. The large deviations in C alpha positions between SET and BT or between SET and SGT are mainly observed in the first domain. The conformations of the side-chains of the catalytic triad are mutually similar to each other in these three proteases. Each of the chi 1 torsion angles of the three residues is distributed within +/- 5 degrees from each corresponding mean value. The hydrogen bond distances related to the side-chains in the triad coincide fairly well, though the relative disposition of the side-chains differs by 0.1-0.6 A among SET, BT, and SGT. The hydrogen bond network concerned with Ser(195), Asp(189), and water molecules in the substrate binding pocket differs from that in BT or SGT.

Cloning and expression of trypsin-like enzyme from Streptomyces fradiae for comparative analysis of functional regions of Streptomyces and mammalian trypsins

The trypsin-like enzyme from Streptomyces fradiae (SFT) is one of the extracellular proteases secreted by gram-positive bacteria. Since the primary structure of SFT is still unknown, a gene encoding SFT was cloned from a S. fradiae genomic library using an amplified polymerase chain reaction product within the SFT gene as a probe. The nucleotide sequence of the cloned fragment revealed that the gene encoded an open reading frame with 259 amino acids. The 38 N-terminal amino acids resemble a typical prokaryotic signal peptide, but the predicted signal sequence cleavage site suggests the existence of a very short, four-amino-acid prosequence. The mature SFT consists of 221 amino acids with a molecular weight of 22900. SFT was expressed in Streptomyces lividans 1326 using pIJ702 as a vector, and the secreted protein was purified from culture supernatant by soybean trypsin inhibitor-affinity chromatography. The N-terminal amino acid sequence and molecular weight of the protein were identical to those of natural SFT, indicating correct processing by S. lividans 1326. Also, the amino acid composition of the recombinant SFT agreed with that of natural SFT and that deduced from the nucleotide sequence. Comparison of the amino acid sequence of SFT and other trypsins of microbial and mammalian origins revealed that SFT exhibits 85% identity to Streptomyces griseus trypsin (SGT), but low identity to Saccharopolyspora erythraea trypsin (SET) (38%) and to bovine trypsin (35%). The sequence alignment shows that the catalytic triad, the substrate-binding site and six cysteine residues are highly conserved. We found amino acid substitutions between SFT and SGT in regions involved in substrate specificity and sequence differences between Streptomyces and mammalian trypsins in substrate-binding regions.

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene

Streptomyces griseus trypsin (E.C.3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.

Crystal Structure of Streptomyces erythraeus Trypsin at 2.7 Å Resolution1

The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 A resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4,968 reflections between 8 and 2.7 A resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Comparison of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 3(10)-helix is found through Ala56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.