Streptomyces Griseus Protease Research Articles

Ruminal Crude Protein Degradation Determined in Sacco and by Co-Incubation of Streptomyces griseus Protease and Carbohydrases.

The objectives of the study were to examine the effect of an antibiotic solution applied in the Streptomyces griseus protease method (SGPM) and the effect of carbohydrases in SGPM on the effective crude protein (CP) degradation (ED) with reference to in sacco ED. For this purpose, the ruminal CP degradation of rapeseed meal, dried distillers' grains with solubles, wheat grain, corn grain, corn silage, grass silage and partial crop field pea silage was determined in sacco using three rumen-fistulated dairy cows and in vitro using SGPM. The impact of the antibiotic solution on CP degradation by S. griseus protease was investigated by supplementing SGPM with Penicillin-Streptomycin solution to reduce microbial mass proliferation during incubation. The carbohydrase α-amylase or Viscozym® L (cell wall-degrading enzyme mixture) was added to the SGPM at four different doses simultaneously as a co-incubation to improve feed protein accessibility. For most feedstuffs, ED was lower when the antibiotic solution was used in SGPM (p < 0.05). The use of an antibiotic solution in the SGPM is recommended to standardize the SGPM. The in sacco ED values were significantly underestimated by the SGPM and by the SGPM with co-incubated carbohydrase (p < 0.05). Co-incubation of S. griseus protease and carbohydrase was not successful in reducing the differences to the in sacco CP degradation.

Comparative Evaluation of Enzymatic Crude Protein Degradation in Selected Legume Forages

For protein evaluation of feedstuffs for ruminants, the Streptomyces griseus protease test offers a purely enzymatic approach to estimate ruminal protein degradation. This study was conducted to determine the enzymatic crude protein (CP) degradability of alfalfa, sainfoin, and common vetch hays, which are commonly used in ruminant nutrition. To estimate CP degradation, fifteen samples from each type of hay were incubated in vitro with a commercial protease extracted from Streptomyces griseus. The incubation was carried out for 1, 4, 24, and 48 hours in a borate-phosphate buffer at pH 8. Significant differences in CP degradability values were found among all three types of hay across all incubation periods. For all incubation periods, sainfoin had the lowest CP degradability values (P &lt; 0.05), due to its high content of cell wall components and condensed tannins (CTs). For incubation periods longer than 1 hour, common vetch had the highest CP degradability values, followed by alfalfa and sainfoin, respectively (P &lt; 0.05). As a result, the use of the protease enzyme extracted from Streptomyces griseus was confirmed as an effective method for estimating the CP degradability of selected legume forages in the laboratory, eliminating the need for animal testing. However, since plant proteins are often embedded within carbohydrate complexes, it is recommended that future tests consider the combined use of protease and carbohydrase, particularly for sainfoin, which is rich in cell wall components and condensed tannins.

The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation.

For protein evaluation of feedstuffs for ruminants, the Streptomyces griseus protease test provides a solely enzymatic method for estimating ruminal protein degradation. Since plant proteins are often structured in carbohydrate complexes, the use of carbohydrase during the test might improve its accuracy. It is advisable to co-incubate protease and carbohydrase, risking that the carbohydrase activity is reduced under the influence of the protease. The present study was conducted to investigate this impact by using α-amylase or the multi-enzyme complex Viscozym® L as carbohydrase. The detection of active protease was determined fluorescence photometrically using internally quenched fluorogenic substrates (IQFS). Cellulose, pectin, and starch degradation were determined spectrophotometrically using 3,5-dinitro salicylic acid as a colorimetric agent. The Streptomyces griseus protease mixture proved to be active for the selected IQFS immediately after the start of measurements (p < 0.05). Starch hydrolysis catalyzed by α-amylase or Viscozym® L, respectively, was decreased by co-incubation with protease mixture by maximal 3% or 37%, respectively, at 5 h incubation time (p > 0.05). Pectin and cellulose hydrolysis catalyzed by Viscozym® L, respectively, was not significantly influenced by co-incubation with a protease mixture (p > 0.05). Although a decrease in carbohydrase activity during co-incubation with Streptomyces griseus protease occurred, it was only numerical and might be counteracted by an adapted carbohydrase activity.

Feed Clusters According to In Situ and In Vitro Ruminal Crude Protein Degradation.

Effective degradation (ED) of crude protein (CP) was estimated in vitro at 0.02, 0.05 and 0.08 h-1 assumed ruminal passage rates for a total of 40 feedstuffs, for which in situ ED was available and used as reference degradation values. For this, the Streptomyces&amp;nbsp;griseus protease test was used. The differences between in vitro CP degradation and the in situ CP degradation values were lowest in legume grains and highest in cereal by-products and barley. The differences between in situ and in vitro ED were expressed using a degradation quotient (degQ), where degQ = (EDin vitro - EDin situ)/EDin situ. Among the tested feedstuffs, eight specific clusters were identified according to degQ for the assumed passage rates. The feedstuffs clustered in an unspecific way, i.e., feedstuffs of different nutrient composition, origin or treatment did not necessarily group together. Formaldehyde-treated rapeseed meal, soybean meal, wheat, a treated lupin, sunflower meal and barley could not be assigned to any of the clusters. Groupwise degradation (range of degQ for assumed passage rates are given in brackets) was detected in grass silages (-0.17, -0.11), cereal by-products together with sugar beet pulp (-0.47, -0.35) and partly in legume grains (-0.14, 0.14). The clustering probably based on different specific nutrient composition and matrix effects that influence the solubility of feed protein and limit the performance of the protease. The matrix can be affected by treatment (chemically, thermally or mechanically), changing the chemical and physical structure of the protein within the plant. The S. griseus protease test had reliable sensitivity to reflect differences between native feedstuffs and treatments (thermally or chemically) that were found in situ. The in situ results, however, are mostly underestimated. The clustering results do not allow a clear conclusion on the groupwise or feed-specific use of carbohydrate-degrading enzymes as pre- or co-inoculants as part of the S. griseus protease test and need to be tested for its potential to make this test more conform with in situ data.

The in vitro protein degradability of legume and sudan grass forage types and ensiled mixtures

This study examined the in vitro crude protein (CP) degradability of wilted, dried for hay and ensiled Medicago sativa, Trifolium pratense and Sorghum sudanense, as well as of ensiled legume-sudan grass mixtures. The rumen degradable protein (RDP) was estimated using the latest Cornell net carbohydrate and protein system (CNCPS v6.5) and with the Streptomyces griseus protease assay (48 h of incubation). Sudan grass forages were the lowest in moderately degradable CP and the highest in CP fraction C, while red clover showed the least values for the soluble true protein like alfalfa forages for slowly degradable protein bound in neutral detergent fibre (NDF). Ensiling of legume-sudan grass mixtures decreases N-NH3 content by 12.2 and 5.1% in comparison with alfalfa and red clover silages, respectively. The RDP values for sudan grass (fresh, wilted, hay) were lower by 7.2 to 7.9% or 10.4 to 15.7% (CNCPS or S. griseus procedure) and up to 0.8 or 5.3 to 9.7% in comparison with alfalfa and red clover, respectively. The reduction of protein degradability in ensiled legume-sudan grass mixtures was 5.5 or 6.1% and 1.5 or 3% compared to alfalfa and red clover silages, respectively. Due to higher rumen undegradable protein content, the sudan grass and legume-sudan grass mixtures may be efficiently used to improve the protein utilization in ruminant nutrition.

Reply to Kumari and Singh, "Antibiofilm Activity of Small Molecules Produced by Staphylococcus epidermidis against Staphylococcus aureus".

We appreciated Kumari and Singh´s comments on our paper (11). Although we have continued trying to determine the identity of the bioactive molecules present in Staphylococcus epidermidis cell-free conditioned media (CFCM), we are yet to be successful. Initially, phenol-soluble modulins (PSMs) were a potential candidate. However, the protease treatment used in our study, which did not abrogate CFCM activity, was somewhat similar to the treatment used by Bojet et al. (1), where supernatant activity due to PSMs was abolished by addition of a Streptomyces griseus protease mixture at 1 mg/ml at 37°C for 30 min followed by heat inactivation (80°C for 20 min). In our study, we treated CFCM with proteinase K (20 mg/ml) for 1 h at 37°C and also performed heat inactivation (100°C for 40 min) without any reduction of antibiofilm activity. In an independent study, Li et al. (2) also abolished supernatant activity due to PSMs by treating it with proteinase K (100 μg/ml) at 37°C for 1 h. Although these conditions are similar to the ones used in our study, it is important to note that the proteinase K concentration employed was 200-fold lower than the one we used and was still sufficient to eliminate PSM activity. Nevertheless, Kumari and Singh are correct when they state that the conformation of PSM aggregates can affect protease susceptibility, and we agree that it might be worth checking the effect of purified S. epidermidis PSMs on Staphylococcus aureus biofilms. Even if PSMs are involved, we believe that, altogether, our data suggest that the bioactivity detected is multifactorial and may involve multiple molecules present in S. epidermidis CFCM. Our transcriptome sequencing (RNA-seq) results showed a huge impact of CFCM on gene expression, repressing the expression of genes associated with both biofilm formation and quorum sensing. These two activities are thought to be inversely regulated, and inhibition of quorum sensing is associated with increased biofilm formation (3–5). Previous work has shown that PSMs from the same species prevent biofilm formation and promote biofilm disruption in both S. aureus and S. epidermidis (6–9). If PSMs prove to be one of the antibiofilm molecules in S. epidermidis CFCM, it will be interesting to determine their mechanism of action and activity during various interspecies interactions, as has been shown by Kumari and Singh regarding Rhizopus arrhizus (10). We thank the authors for all the suggestions for experiments, and we believe that testing CFCM of an agr mutant strain of S. epidermidis will be a valuable addition to future work.

Effect of toasting grain silages from field peas (Pisum sativum) and field beans (Vicia faba) on invitro gas production, methane production, and post-ruminal crude protein content.

Legume grains such as field peas and field beans can be produced on a local level, and may be reliable sources of dietary protein and energy apart from common soybean and rapeseed meals. In ruminants, protein, starch, and carbohydrates from peas and field beans are fermented in large part before reaching the small intestine. The objective of this study was to evaluate the effects of a combination of ensiling and hydro-thermic treatment (i.e., toasting at 160 °C for 30 min) of grains of peas and field beans on the concentrations of post-ruminal crude protein (PRCP) and rumen-undegraded protein (RUP). Moreover, 24-h gas production and methane production were measured. For this, an in vitro batch culture system with ruminal fluid from sheep was used. Rumen-undegraded protein was determined using the Streptomyces griseus protease test. Scanning electron micrographs were used to visualize morphological changes of starch granules and their joint matrices in peas and field beans after ensiling, toasting, or a combination of both. Native pea grains contained crude protein (CP) at 199 g/kg DM, PRCP at 155 g/kg DM at a ruminal passage rate of 0.08/h (Kp8), RUP at 33 g/kg DM at Kp8, and starch at 530 g/kg DM. Native field beans contained CP at 296 g/kg DM, PRCP at 212 g/kg DM at Kp8, RUP at 54 g of/kg DM at Kp8, and starch at 450 g/kg DM. The PRCP did not considerably differ among native and treated peas or field beans. Especially in the peas, RUP at Kp8 increased after ensiling by 10 g/kg DM (i.e., 30%; P < 0.05). Toasting increased RUP (Kp8) in ensiled peas by another 28% (P < 0.05). Toasting had no effect on PRCP or RUP when the peas or field beans were not ensiled before. Gas and methane production were not affected by any treatment, and scanning electron micrographs did not reveal structural changes on the starches doubtless of any treatment. Protein seemed to be more affected by treatment with ensiled + toasted peas than with ensiled + toasted field beans, but starches and other carbohydrates from both legumes remained unaffected.

Effects of toasting temperature and duration on in vitro ruminal gas production kinetics and post-ruminal crude protein from field pea (Pisum sativum) legume grain silages

Field peas provide valuable starch and protein, but especially the latter is highly soluble in the rumen. Toasting of ensiled pea grains might reduce ruminal starch and protein degradation. The objective was to test this by measuring 24 h total gas production, gas production kinetics, and post-ruminal crude protein (PRCP) in an in vitro batch culture system. First, toasting temperature was varied with 120, 140, 160, 180, and 200 °C stages, while keeping a constant treatment duration of 30 min, and second, toasting temperature was kept constant at 160 °C, and the duration was varied with 10, 20, and 30 min. Rumen-undegraded protein (RUP) was measured using the Streptomyces griseus protease test. Moreover, ensiling and toasting effects on the morphology of pea starches were visually examined using scanning electron microscopy. In vitro gas production decreased with increasing toasting temperature or duration of heat exposure (P < 0.05). Morphological changes of starch granules and embedding matrices became visible at a toasting temperature of at least 180 °C. Post-ruminal crude protein was less affected by ensiling and ensiling + toasting. However, ensiling + toasting decreased the amount of soluble protein, but increased the insoluble protein fractions. Furthermore, protein solubility decreased with increasing temperature (by at most 41% of crude protein) or duration of toasting (by at most 21% of crude protein), and RUP increased by approximately 10 g/kg dry matter (DM) through ensiling, and by maximally 40 to 50 g/kg DM through toasting (P < 0.05). The results indicate that ensiling and subsequent toasting of field peas might elevate the concentration of ruminally stable carbohydrates and protein, which, however, do not necessarily result in elevated PRCP concentrations. Whether we achieve protein protection or protein damage after heat exposure depends on the temperature and duration of the toasting process.

Ruminal In Vitro Protein Degradation and Apparent Digestibility of Energy and Nutrients in Sheep Fed Native or Ensiled + Toasted Pea (Pisum sativum) Grains.

Simple SummaryPea grains may partially replace soybean or rapeseed meals and cereals in ruminant diets, but this is limited by high solubility of pea protein in the rumen. Hydro-thermic treatments such as toasting may stabilize the protein and shift digestion from the rumen to the small intestine. The effect of toasting of ensiled pea grains on rumen-undegraded protein was tested in vitro and on apparent digestibility of organic matter, gross energy, and proximate nutrients in a digestion trial with sheep. Ensiling plus toasting increased rumen-undegraded protein from 20 to 62% of crude protein, but it also increased acid detergent insoluble protein, which is unavailable for digestive enzymes in the small intestine from 0.5 to 2.6% of crude protein. Ensiling plus toasting did not, however, affect total tract apparent digestibility of organic matter, energy, crude protein, or any other nutrient fraction, nor did it alter the concentration of metabolizable energy or net energy lactation in the peas. The technique can be implemented on farms and might have a positive impact on field pea production.Pea grains may partially replace soybean or rapeseed meals and cereals in ruminant diets, but substitution by unprocessed peas is limited by high ruminal protein solubility. The effect of combined ensiling and toasting of peas using a mobile toaster (100 kg/h throughput rate, 180 to 190 °C supplied air temperature) on rumen-undegraded protein (RUP) was tested in vitro using the Streptomyces griseus protease test. The effects of ensiling plus toasting on apparent digestibility of organic matter (OM), gross energy (GE), and proximate nutrients were examined in a digestion trial. Concentrations of metabolizable energy (ME) and net energy lactation (NEL) were calculated. Native peas had 38 g RUP/kg dry matter (DM), which was 20% of crude protein (CP). Rumen-undegraded protein increased three-fold after ensiling plus toasting (p < 0.001). Acid detergent insoluble protein increased five-fold. Apparent digestibility was 0.94 (OM), 0.90 (CP), and above 0.99 (nitrogen-free extract, starch, and sugars) and was not altered by the treatment. The ME (13.9 MJ/kg DM) or the NEL (8.9 MJ/kg DM) concentration was similar in native and ensiled plus toasted peas. This technique can easily be applied on farms and may increase RUP. However, it needs to be clarified under which conditions pea protein will be damaged.

Protein degradability of grassland forage under simulated rotational spring grazing

A cutting experiment was conducted to analyze the changes in the crude protein (CP) fraction content and in the estimated ruminal protein degradability of forage, obtained in conditions of simulated rotational spring grazing on permanent grassland. The field trial was conducted on permanent pasture during 2015 and included three cuttings as a simulated rotational spring grazing. For determination of protein degradability of pasture forage, the fractionation of the CP according to Cornell Net Carbohydrate and Protein System (CNCPS v6.5) and the Streptomyces griseus protease assay were used. Relative to CP, no significant differences were found among cuts for ammonia N content (A1 fraction) and for protein fraction C which is completely unavailable to the animals. Values for soluble true protein (A2 fraction) and cell wall-associated protein, which is acid detergent soluble (B2), were significantly increased (p&lt;0.05) while a significant reduction (p&lt;0.05) of the moderately degradable protein (B1) content was determined during the growing season. The lower rumen degradable protein (RDP) content of grassland herbage was obtained in the second cut which was significant (p&lt;0.05) according to the CNCPS procedure. Obtained high solubility and degradability of CP in pasture require adequate content of readily available carbohydrates in rations for grazing ruminants to provide efficient utilization of consumed protein.

Determination of protein degradability of alfalfa hay via buffer or protease

This study was conducted to determine the effect of different vegetative periods on protein fractions of alfalfa hay and to compare two different methods for estimation of its protein degradability. In this study, 44 alfalfa hay samples cut in late vegetative, late bud, early bloom and late bloom were used. Crude protein decreased with advancing maturity (p < .05), but neutral detergent insoluble nitrogen and acid detergent insoluble nitrogen were similar. Protein degradability of the samples was estimated using Streptomyces griseus protease or borate–phosphate buffer, kinetic model, as described in Cornell Net Carbohydrate Protein System (CNCPS). Alfalfa hay samples were subjected to proteolysis for 30 h with 0.115 U/mL or 0.230 U/mL of the protease at pH 6.8. There was a moderate correlation (0.66, 0.72) between enzymatic and buffer method estimates with protein degradation. As vegetation progresses, rapidly degradable fraction (A) decreased (p < .05) and undegradable fraction (C) increased (p < .05), while potentially degradable fraction (B) was unchanged. Rumen degradable protein content decreased (p < .05) with advancing maturity. The enzyme/duration limits should be standardised and investigated whether the protease enzyme alone is sufficient for all forages.

Nitrogen fractionation of certain conventional- and lesser-known by-products for ruminants.

Dietary proteins for ruminants are fractionated according to solubility, degradability and digestibility. In the present experiment, 11 vegetable protein meals and cakes used in ruminant nutrition were included with a main focus on determining various nitrogen (N) fractions in vitro. Total N (N × 6.25) content varied from 22.98% (mahua cake) to 65.16% (maize gluten meal), respectively. Guar meal korma contained the lowest and rice gluten meal had the highest acid detergent insoluble nitrogen (ADIN; N × 6.25). Borate-phosphate insoluble N (BIN, N × 6.25) and Streptomyces griseus protease insoluble N (PIN; N × 6.25) were higher (P < 0.01) in maize gluten meal than in other feeds, whereas groundnut cake and sunflower cake had lower (P < 0.01) BIN, and PIN, respectively. Available N, calculated with the assumption that ADIN is indigestible, was maximum in guar meal korma and minimum in rice gluten meal. Furthermore, rapid and slowly degradable N (N × 6.25) was found to be higher (P < 0.01) in groundnut cake and coconut cake, respectively. Intestinal digestion of rumen undegradable protein, expressed as percent of PIN, was maximum in guar meal korma and minimum in rice gluten meal. It was concluded that vegetable protein meals differed considerably in N fractions, and therefore, a selective inclusion of particular ingredient is needed to achieve desired level of N fractions to aid precision N rationing for an improved production performance of ruminants.

Specificity of Proteinase K at P&lt;sub&gt;2&lt;/sub&gt; to P&lt;sub&gt;3&lt;/sub&gt;’ Sub-sites and its Comparison to other Serine Proteases

Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

Functional properties of protein hydrolysates from pea (Pisum sativum, L) seeds

SummaryThe aim of this study was to investigate the effects of partial enzymatic hydrolysis on functional properties of two different pea protein isolates obtained from two pea genotypes, Maja and L1. Papain and commercial protease (Streptomyces griseus protease) were used for protein modification. Solubility, emulsifying and foaming properties were estimated at four different pH values (3.0, 5.0, 7.0 and 8.0). Papain increased solubility of L1 pea protein isolate at pH 3.0, 5.0 and 8.0, emulsifying properties and foaming capacity at all pH values. Otherwise, papain increased solubility of Maja pea protein isolate only at pH 8.0. This pea protein isolate modified with both enzymes formed emulsions with improved stability at lower pH (3.0, 5.0). The commercial protease‐prepared pea protein isolates showed generally low solubility and different emulsifying and foaming properties. Proper selection of enzyme, conditions of hydrolysis and genotypes could result in production of pea protein isolates with desirable functional properties.

Forage management effects on protein and fiber fractions, protein degradability, and dry matter yield of red clover conserved as silage

Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover ( Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein (CP) and fiber fractions in red clover silage and dry matter (DM) yields during the first full production year. In 2003 and 2006, red clover was cut early on 7 June or late on 14 June with two ∼40 d regrowth cuts. For comparison, alfalfa ( Medicago sativa L.) was cut early on 26 May with three ∼32 d regrowth cuts or late using the same schedule as early cut red clover. After ensiling at 370 g/kg DM, CP and fiber were fractionated according to the Cornell Net Carbohydrate and Protein System (CNCPS). RDP and RUP were calculated from CNCPS fractions or estimated by a more convenient Streptomyces griseus protease procedure, which gave highly related ( R 2 ≥ 0.95) estimates of CP degradability. Early cutting of red clover favorably decreased RUP, neutral detergent fiber (NDF), and acid detergent fiber (ADF) and increased RDP and CP in the first two harvests. Regression analyses indicated desirable levels of RDP (>150 g/kg DM) and NDF (∼400 g/kg DM) could be obtained by harvesting red clover at a mean stage weight maturity of 2.0 at first cut, 3.0 at second cut, and 3.5–4.0 at third cut. Averaged across harvest schedules, red clover had lower RDP, CP and ADF, higher RUP, and similar NDF compared to alfalfa. Total DM yields of red clover and alfalfa were not influenced by harvest schedule. Time of establishment had little effect on silage quality, but it did influence total DM yields; April vs. August seeding depressed red clover yields (12.3 t/ha vs. 13.1 t/ha) but enhanced alfalfa yields (12.5 t/ha vs. 11.9 t/ha) during the first full production year. Overall, these results suggest protein utilization and performance of dairy cattle could be moderately improved without sacrificing DM yield by taking a very early first cutting of red clover followed by regrowth cuttings made at progressively later maturities.

Chronic Administration of Lipopolysaccharide and Proteases Induces Periodontal Inflammation and Hepatic Steatosis in Rats

Epidemiologic studies suggest a relationship between periodontitis and liver diseases. A rat periodontitis model was used to investigate whether a causal relationship exists between periodontitis and liver diseases. Fourteen male Wistar rats (8 weeks old) were divided into two groups: a periodontitis group in which Escherichia coli lipopolysaccharide (LPS) and Streptomyces griseus proteases were applied into the gingival sulcus for 8 weeks, and a control group using pyrogen-free water instead. After blood samples were collected, periodontal tissues and liver specimens were analyzed. Chronic administration of LPS and proteases to the gingival sulcus induced periodontitis and liver injury, including steatosis with inflammation and sinusoidal fibrosis. Apoptosis, enhanced concentration of 8-hydroxydeoxyguanosine, and activated production of tumor necrosis factor-alpha in liver were observed in the periodontitis group, with increased gingival inflammation, serum LPS, and reactive oxygen species. Periodontal inflammation in a rat model induced fatty liver disease through increased serum LPS.

Crystal Structures of Five Bovine Chymotrypsin Complexes with P1 BPTI Variants

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.

Generation of a thermostable and denaturant-resistant peptide ligase.

The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.

Caracterização de hidrolisados enzimáticos de pescado

Hidrolisados enzimaticos sao utilizados no tratamento clinico de pacientes com dificuldade em digerir e absorver proteinas, sendo preferidos em relacao as misturas de aminoacidos livres. Neste trabalho foram caracterizados quimicamente seis hidrolisados de minced de pescado, obtidos pelo emprego de diferentes sistemas enzimaticos, quanto a extensao da hidrolise, distribuicao de peso molecular dos peptidios, composicao quimica e perfil de aminoacidos. A hidrolise resultou na solubilizacao de 63,4 a 94,2% das proteinas, sendo esta diretamente proporcional ao grau de hidrolise e dependente do sistema enzimatico e das variaveis do processo (relacao enzima/substrato e atividade enzimatica). A composicao dos hidrolisados atendeu a recomendacao de ingestao dietetica para aminoacidos essenciais, tanto para criancas como para adultos. A relacao entre a concentracao de aminoacidos ramificados e aromaticos (Relacao de Fischer) resultou em valores superiores a 3,5 indicando que os hidrolisados obtidos podem ser uteis para a manutencao dietetica de pacientes com doencas hepaticas cronicas. Os hidrolisados forneceram peptidios com pesos moleculares bem definidos, destacando-se o obtido com pepsina e protease de Streptomyces griseus, que apresentou 57% de peptidios menores do que 3 kDa, adequado para o uso em formulacoes hipoalergenicas.

The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability.

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and alpha-lytic protease (alphaLP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of alphaLP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and alphaLP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and alphaLP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ~10(5) in the spontaneous folding rate and ~5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.