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An essay was developed to measure the activity of the enzyme L-cysteine desulfhydrase in an agricultural loam soil. Linearity was demonstrated between CDA and a length of incubation (up to 3.5hrs),b) the amount of soil used (up to 0.75g),and the concentration of L-cysteine(up to 1.8mM). These demonstrated lineararities show that the assay method employed measures the hydrolysis of L-cysteine and that the measured enzyme activity is not limited by any of the parameters employed. The optimum temperature for CDA in this soil was 55°C and the optimum pH for CDA in soils was pH 8.5. Cysteine desulfhydrase in the soil required the addition of pyridoxal phosphate to exhibit its maximum activity. However since no pyridoxal phosphate was found in this soil it is likely that activity of this enzyme in the soil will be limited by the lack of this cofactor. Our studies illustrate the important point that not all enzymes which can be assayed in soils under laboratory conditions will function in the environment, since some, as in the case of CDA will be limited by a lack of an essential co-factor.
Key words: L-cysteine, enzyme, bacteria, fungi, environmental microbiology, soil.
Soils are known to exhibit a wide range of enzyme activities which are important in the cycling of nutrients in the carbon, nitrogen and sulphur cycles (Skujins, 1976 and Burns, 1979). These enzymes are derived from soil animals, plants and microorganisms and are often immobilised in soils. Enzyme activity has been used to measure overall microbial activity(e.g. dehydrogenase activity) and the activity of microorganisms involved in more specific soil transformations, such as urea hydrolysis (urease) and the breakdown of cellulose (cellulase) (Burns,1979 and Burns,1982). With the exception of the measurement of arylsulfatase activity (Li and Sarah, 2003) relatively little attention has been paid to the soil enzymes involved in the mineralisation of organic sulphur compounds. Here, we report on the measurement of cysteine desulfhydrase activity (CDA) in an agricultural soil. This enzyme is important in the degradation of the amino acid cysteine, and therefore of organic sulphur, because it catalyses the desulfhydration of cysteine, liberating equimolar quantities of pryruvate, hydrogen sulphide and ammonia. The enzyme is widely distributed in bacteria and fungi (Okishi et al.,1981) and although it has been reported to occur in coastal sand dunes (Skiba and Wainwright, 1983), its activity in a fertile agricultural soil, like one used here, appears not to have been previously reported. Here we report on CDA activity in soil by measuring the formation of pyruvate when L-cysteine and pyridoxal sulphate were incubated with soil in the presence of buffer. The following questions were asked a) can CDA be assayed in soil and b) if so, does this enzyme function in the soil environment?
Material and Methods
An agricultural sandy loam (previous crop potatoes, was used (organic C, 3.2%; total N, 0.3%; pH, 6.1).
Triplicate samples of field moist soil (1g) were treated with toluene (0.5ml) in universal bottles closed with screw caps and left for 15 min. The enzyme reaction was started by adding Tris HCl buffer (3 ml,0.2M, pH 8.3), L-cysteine (5mM, 1ml) and pyridoxal phosphate, 0.2mM, 1 ml),both dissolved in Tris HCL buffer, 2mM, pH ,8.3). The contents of the bottles were thoroughly mixed and the bottles were incubated at 37°C. After 2hrs the enzyme reaction was stopped by adding trichloroacetic acid (TCA 10% w/v).Two sets of controls were included a) where no cysteine was added and b) where the enzyme reaction was stopped immediately. The soils suspensions were filtered through Whatman No.1 filter paper and CDA activity was measured by the determining the concentration of pyruvate as follows.
Determination of pyruvate
To the filtrate (0.5 ml) was added TCA, 0.3ml 0f a 50% w/v solution) distilled water (2.2 ml) and 2,4-dintrophenylhydrazine (1 ml, of a 1% solution in 2M HCl); mixed and left for 10min at room temperature. Sodium hydroxide (5 ml of a 2.5N solution) was then added and after 10min incubation at room temperature, the reddish brown colour formed was measured at 445nm. The pyruvate concentration was then determined by reference to a standard curve ranging from 0-0.1µ moles pyruvate.
Development of the CDA assay
By using the basic assay described above and varying one parameter at a time, the optimum conditions for the assay of CDA in this soil was determined. The following were determined; a) the optimum amount of soil; the reaction mixture was incubated for 2hrs at 37°C with one of the following 0, 0.5, 1.0, 2.0, 4.0 g of soil; b) period of incubation the reaction mixture was incubated at 37°C for 0, 2, 5, 10, 15, 25 hrs; c) substrate concentration, L-cysteine concentration of 0, 0.4, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0). Effect of temperature on CDA reaction mixtures were incubated for 2hrs at 10, 20, 25, 40, 50, 60, and 80°C) effect of pH CDA was assayed using Tris-HCL buffer over the following pH range pH 7.0, 7.5, 8.0., 8.5, 9.0, 10.0.
Determination of pyridoxal phosphate in soil
Soil (10 g) was shaken for a period of 1hr with Tris-HCl buffer (0.2 M, pH 8.3, 100 ml) and then filtered through a Whatman No. 1 filter paper. The concentration of pyridoxal phosphate in the filtrate was then determined by the following method (Wada and Snell, 1961). Phenlyhyrdazine hydrochloride (0.2 ml, 2% w/v dissolved in 10 N H2SO4) was added 3.8 ml of soil filtrate. The mixture was then heated at 60°C for 20 min and then allowed to stand at room temperature for 10 min and when the intensity of the colour formed was read at 410 nm.
Linearity was demonstrated between CDA and a) length of incubation (up to 3.5hrs.) b) the amount of soil used (up to 0.75g) and the concentration of L-cysteine (up to 1.8 mM) (Figs: I a, b, c). These demonstrated lineararities show that the assay method employed measures the hydrolysis of L-cysteine and that the measured enzyme activity is not limited by any of the parameters employed.
Fig: I. Effect of a) soil sample b) time and c) substrate concentration on L-cysteine desulfhydrase activity in the soil.
The optimum temperature for CDA in this soil was 55°C (Fig: II a) which is higher than that reported by Fromageot (1951) for the enzyme in bacteria and mammals. Soil enzymes generally show a higher temperature optimum than is seen for pure enzymes, or when enzyme activity is measure in cells; this is because soil enzymes are immobilized onto clays and humus particles. The optimum pH for CDA in soils was pH 8.5 (Fig: 2b). This pH optimum is the same as that found for Salmonella typhimirium (Guarneros and Ortega, 1970), but somewhat higher than that found in other bacteria (Fromageot,1951) again because of soil immobilization soils; enzymes often show broader and higher pH maximum than enzyme activities measured in other systems .
Fig: II . Effect of (a) incubation temperature and (b) buffer pH on L-cysteine desulfhydrase activity in the soil.
Table-I shows and important property of CDA, namely that it requires the presence of pyridoxal phosphate to exhibit its maximum activity. Stimulation of CDA by pyridoxal phosphate was also reported for this enzyme from Proteus morganii (Kallio, 1951) and E.coli (Delwiche, 1951). (Table-I)
Table 1 Effect of pyridoxal phosphate on CDA in soil
CDA µg pyruvate formed g-1 2h-1
No pyridoxal phosphate added 0.080 (0.004)
Addition of pyridoxal phosphate (1ml, 0.2M). 0.295 (0.015)
Means of triplicates (+/- standard deviation).
Pyridoxal phosphate was not detected in this agricultural soil (1:10 w/v soil 0.2M Tris-HCL buffer (pH8.3), extracted by shaking for 1h.), showing that either pyridoxal phosphate is not extracted by the method, or more probably that it is not present in detectable concentrations in this soil. The lack of pyridoxal phosphate in this soil means that CDA in to operate in this (and presumably other soils) at its maximum activity because of the lack of a necessary cofactor, namely pyridoxal phosphate.
The low level of pyridoxal phosphate (which is a necessary cofactor required for optimal functioning of CDA) in this soil used suggests that CDA will not function in this and other soils, at least to any high level of activity. Burns (1979) emphasised that cytoplasmic enzymes from animals, plants
and microorganism, which rely upon co-factors, electron transport chains or multi enzyme complexes will not operate in soils unless such cofactors are present. L-Cysteine desulfhydrase provides an excellent example of an enzyme which is present is soil, probably bound to humus and clay particles which, because of a lack of necessary cofactors cannot function in vivo. Activity of the enzyme can however, be measured in vitro when the necessary cofactors (in this case pyridoxal phosphate) is added. The present study therefore illustrates the important point that because an enzyme can be assayed in soil it does not necessarily show that it can function in the environment. Enzymes such as cellulase (Benefield, 1971), urease (Bremner and Mulvaney,1978) and o-phenol oxidase (Wainwright, 1979), on the other hand, which do not require cofactors will likely function in the environment. The important fact that the fact that an enzyme can assayed in the environment without it necessarily playing an important role in mineral cycling is the main conclusion of this work.
Thanks are due to Dr Ute Skiba for her contribution to this study. The study was supported in part by the Centre for Excellence and Diversity, King Saud University; we also thank the College of Science Research Center, King Saud University, Saudi Arabia, for support.