A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization

A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.     

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae)

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.     

Proteolytic activities in dormant rye (Secale cereale L.) grain.

Endoproteolytic, exoproteolytic, carboxypeptidase, aminopeptidase, and N-alpha-benzoyl-arginine-p-nitroanilide hydrolyzing activities were detected in 0.05 M sodium acetate buffer (pH 5.0) extracts of whole meal of the rye (Secale cereale L.) varieties Amando, Halo, and Humbolt. The proteolytic enzymes of Humbolt, the variety with the highest proteolytic activity, optimally hydrolyzed hemoglobin around pH 3.5 and 40-45 degrees C. In the different milling fractions of Humbolt, azocasein and hemoglobin hydrolytic activities were especially found in the bran and shorts. Proteolytic enzymes in the bran extract were concentrated in the 35-60% ammonium sulfate precipitate. Pepstatin A, an inhibitor of aspartic proteases, reduced approximately 88 and approximately 75% of the hemoglobin and azocasein hydrolyzing activities of this precipitate, respectively. Phenylmethanesulfonyl fluoride, an inhibitor of serine proteases, inhibited approximately 33% of both cited activities. Both rye and wheat storage proteins were degraded by Humbolt rye whole meal enzyme extract and the above-mentioned ammonium sulfate rye bran fraction in vitro. With the latter fraction digestion was more pronounced.     

Degradation of gluten by proteases from dry and germinating wheat (Triticum durum) seeds: An in vitro approach to storage protein mobilization

Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.     

Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans

Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Modeling the kinetics of whey protein hydrolysis brought about by enzymes from Cynara cardunculus

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.     

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.     

Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.     

A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.     

Effect of pH on the solubilization of brewers' spent grain by microbial carbohydrases and proteases

The potential for enzymatic solubilization of brewers' spent grain by carbohydrases and proteases was examined over a broad pH range (pH 3.2-11.2). Enzymes from Trichoderma (Depol 686) were most efficient at a lower pH, while enzymes from the Humicola preparation (Depol 740) were the best performer over the whole range. Profiling of key glycoside hydrolase, esterase and protease activities across the pH range demonstrated that solubilization of spent grain by the Trichoderma enzymes corresponded to the range of maximum activities. This was not the case with the Humicola enzymes, where maximum solubilization of the substrate occurred at pH 9.1, at which pH the determined activities were low. Protease activity in Depol 740 was associated with a high solubilization, but inhibition of proteolytic activity resulted in only a 5% decrease in spent grain solubilization. These results suggest that while enzymes can be used to exploit agro-industrials byproduct, the use of high pH increases the extent of hydrolysis and an unidentified factor produced by Humicola improves the enzyme-catalyzed solubilization of lignocellulosic material.     

Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.     

Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV)

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.     

Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15)

Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.     

Benoxacor induction of terbuthylazine detoxification in Zea mays and Festuca arundinacea

The action of safener benoxacor on the detoxification of terbuthylazine (TBA) in Zea mays and Festuca arundinacea was ascertained by the investigation of the effects of benoxacor on the activity of glutathione-S-transferases (GSTs) in the shoots of the two plant species. TBA treatment generally reduced GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) in corn and did not affect the enzyme activity in festuca. When applied alone, benoxacor stimulated GST activity in both plants; however, when it was applied in mixture with TBA, generally, an enhancement of the enzyme activity was found in corn but not in festuca in comparison with the respective TBA-treated samples. The enhancement of GST activity in response to the benoxacor treatment in both corn and festuca resulted to be concomitant with decreases in apparent K(M) in both the plant species, with V(max) unaffected, and with an increased expression of proteins having molecular masses in the characteristic range of plant GSTs. After the benoxacor treatment, increased GST activity toward TBA as a substrate was observed in both corn and festuca. As a consequence, lesser amounts and persistence of TBA residues were found in shoots of corn and festuca treated with the TBA and benoxacor mixture compared to TBA-only-treated samples. Therefore, benoxacor enhances TBA detoxification in both corn and festuca; the induction of detoxifying activity in a nondomesticated grass is discussed in view of its use in vegetating buffer strips around crops to prevent TBA pollution.     

The role of cytokinins,ethephon, and chlorocholine chloride in the native proteolytic activity of forest soils

Purpose

Plant growth-promoting rhizobacteria (PGPRs) synthesizes and exports phytohormones which are called plant growth regulators (PGRs). These PGRs may play a regulatory role in plant growth and development. PGRs are organic substances that influence physiological processes of plants at extremely low concentrations. The objective of this study was to find out whether three PGRs, cytokinins, ethephon, and chlorocholine, have a stimulatory or inhibitory effect on the activity of native proteases in soil.

Materials and methods

A revised methodology was used to determine soil protease activity, where TRIS-HCl buffer was replaced with demineralized water. This is described as native protease activity as its activity is not affected by chemicals. The aim was to approximate the protease response to cytokinins in the native soil environment. The native soil proteolytic activity was determined spectrophotometrically.

Results and discussion

The present paper shows that cytokinin, ethephon, and chlorocholine chloride negatively affect the native proteolytic activity of forest soils with the exceptions of the organic and organomineral horizons of European beech (Fagus sylvatica L.) on a rendzic Leptosol. In addition, 6-benzylaminopurine stimulates the native proteolytic activity of the organic horizon of pedunculate oak (Quercus robur L.). A negative effect of cytokinins on the soil proteolytic activity can decrease the rate of organic matter decomposition. The results provide soil biochemists with an insight into the roles of rhizospheric substances on soil microbial activity.

Conclusions

This work has shown that cytokinins and PGRs inhibit the activity of native soil proteases in most of the studied forest sites. Results describe the effect of rhizospheric compounds on the activity of soil microorganisms, with potentially significant implications for the nitrogen cycle in forest soils.