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Proteases are also known as proteinases or proteolytic enzyme. In enzyme classification, proteases fall in group hydrolases. This class of enzyme catalyses cleavage reactions or reverse fragment condensations. A distinction is made between peptidases, esterases, lipases, glycosidases, phosphatases and so on according to the type of bond being cleaved. The cleavage requires the participation of water molecule.
Proteases act by hydrolyzing peptide bond of long protein chains into short fragments which are amino acid residues. There are 2 types of reactions involved in the cleavage of protein chain which are endopeptidases and exopeptidases. Endopeptidases allow splitting of peptide bond at internal linkage of amino acid residues such as trypsin which cleaves the bond after arginine or lysine amino acids while chymotrypsin cleaves after bond of phenylalanine, tryptophan and tyrosine unless the both of their following amino acid is proline. The optimum pH of these enzyme is pH8. However, exopeptidases allow cleavage of terminal amino acids in protein chain such as aminopeptidase which is present and secreted by glands of small intestine that cuts the bond from the aminoterminal while carboxypeptidase is present in pancreatic juice and functions by cleaving the bond at carboxylic end of peptide.
2.1.2 Sources of proteases
Generally, proteases can be found naturally in human bodies, microorganisms and also plants. (Joanitti et al., 2006; Lawrence and Koundal, 2002).They are present in all living systems since protein is crucial in development and functioning of life. Proteases play important role in human digestive system. They are present as pepsin in stomach and as trypsin or chymotrypsin in duodenum. Pepsin allows digestion of protein in food into small polypeptides. Then, these small polypeptides will be broken down into smaller polypeptides and further digestion into individual amino acids occurs in the small intestine by peptidases.
In our dynamic ecosystem, bacteria play a major role in decomposing dead matters. Saprobic bacteria are responsible as initial bacteria to decompose muscles by degrading the muscle tissue into strands of protein. Due to the ubiquitous microorganisms present in the soil, these protein strands will be broken down into amino acids rapidly by fermentative bacteria.
As in plants, proteases has been identified in pineapple, papaya and fig tree which are bromelain, papain and ficin (unpublished work) respectively (Shukor et al; 2006, 2007, 2008) Bradford-protease-casein system as the principal assay. Proteases that present in plants also have been studied to have a role in defense mechanism from pathogens and pests effect such as soybean, tobacco, and Arabidopsis (Hoorn, 2004). Some of proteases in those plants show defense responses towards invading organisms (Pechan et al.,2002)
Garlic (Allium sativum) is a member of the onion family (Alliaceae) along with onions, chives, shallots, leeks, and elephant garlic. Studies on garlic proposed that it has medicinal value of antibacterial and antiseptic properties. Besides, it has been cultivated for centuries and valued as food as well flavor in cooking. Garlic was reported to have large concentrations of sulfur compounds which contribute to its remedial and natural activities (Abdullah et al., 1988)
Figure 1: Garlic (Allium sativum). a) Garlic bulb. b) Peeled garlic.
2.3 Heavy metals
2.3.1 Definition of heavy metals
Heavy metals are basically inorganic elements those have high relative atomic mass and possess metallic properties. Generally there are approximately 65 elements of them and the specific gravity of these heavy metals is greater than 6g/cm3 (Passaw, 1961). A few examples of these heavy metals are cadmium, mercury, lead, zinc, arsenic, copper, chromium, nickel, silver, and cobalt. Some of them are poisonous to human and persistent in our environment since they cannot be degraded or destroyed. Hence, excessive levels of heavy metals may cause harm towards the environment and human health.
2.3.2 Sources and distributions of heavy metal
Heavy metals do exist naturally in our ecosystem but varies in their concentrations. They are the natural constituents of our earth's crust. Since they are neither degraded nor created, they tend to present in freshwater, seawater, soils and also sediments. In natural, heavy metals may exist as compound or uncombined form. They exert their toxic effects by combining with one or more reactive groups (ligands) essential for normal physiological functions.
Recent years, a study proposed that heavy metal of mercury was geological origin of volcanic activity while others such as chromium, copper, nickel and zinc were natural heavy metals in soil profiles (Dœlscha et al., 2005). Besides, natural disasters also contribute to the distribution of heavy metals throughout the ecosystem. Flood or tsunami accounts for almost 40 percents of overall global natural disasters (Euripidou et al.,2004). This disaster cause massive mobilization or remobilization of heavy metals distribution from sites of agricultural, sewage system, mining and industrial into waterways like rivers, lakes and coastal waters (French et al.,1989).
2.3.3 Heavy metals pollution
Metals are probably the oldest toxins known to humans. Lead usage may have begun prior to 2000 BC in the smelting of silver. Arsenic was obtained during the melting of copper and tin, and an early use was for decoration in Egyptian tombs. For the past few decades, human activity and the development have contributed to elevated pollution level in the environment. However, the level of contamination has reached a certain point that it is no longer an issue of whether one has been exposed to heavy metals, but rather the level of exposure.
Human activity has been recognized to affect the natural distribution of heavy metals into the environment by altering their chemical form. Most of human activities like industrial and agricultural contribute to pollution to waterways due to the release of metal pollutants such as pesticides. Ore processing, smelting and refining operations can cause deposition of large quantities of trace metals, such as lead (Pb), zinc (Zn), copper (Cu), arsenic (As) and silver (Ag), into drainage basins or direct discharge into aquatic environments. Besides that, domestic wastewater effluents also contribute to the heavy metals pollution. It contain large amounts of trace metals from metabolic waste products, corrosion of water pipes such as copper, lead, zinc and cadmium; and household products such as detergents that contain iron, manganese, chromium, nickel, cobalt, zinc, boron and arsenic. Domestic wastewater and the dumping of domestic and industrial sludge are the major artificial sources of cadmium, chromium, lead, copper, iron and mercury pollution.
Digging a mine, removing ore from it, and extraction and processing of the minerals sometimes cause environmental damage and pollute waterways via toxic drainage (Csuros, 2002). High concentrations of heavy metals near highly populated areas gave evidences that human activities and uncontrolled discharge of waste were contributing metal pollution into water (Demirak et al.,2006). Over the past century, industries discharged various types of pollutants into their wastewater causing pollution to our waterways including heavy metals. These heavy metals will eventually be deposited as sediments in aquatic environment which play important roles in mobility and storage of toxic heavy metals (Dang and Jeffrey,2006).
Figure 2: Water quality status of a rivers in Peninsular Malaysia (2006). Source of DOE.
2.3.4 Toxicity of heavy metals
Toxicity can be described as the degree of toxic substances to exhibit its poisonous effects toward biological systems. Toxic substances or toxins are present almost everywhere in our environment. Beneficial minerals or vitamins in our diet might as well become toxin if they were excessively consumed. Heavy metals are one of the global considerations for its toxic characteristics even at very low concentration. The toxicity of heavy metals is not only evaluated for its concentrations but also by their bioavailability (Fjällborg and Dave, 2003). Levels of exposure to heavy metals and it effects also can be varying. It is acute, chronic and lethal effects. Acute effects are symptoms that exert right after exposure and more severe in term of their harmful effects. These effects are generally caused by fairly high concentrations of heavy metals during a short exposure period. There are also chronic effects, which mean it affects are delayed, but long-lasting, responses to the heavy metals. They may occur months to years after exposure and usually persist for year. Generally, this is a result of low-level exposure over a long period. Lethal effects can be defined as responses that occur when physical or chemical agents interfere with cellular and sub cellular processes in the organism to such an extent that death directly follows. Several methods have been developed in order to measure heavy metals toxicity. However a few of them were frequently used such as enzymatic and nitrification inhibition, effluent turbidity, and oxygen uptake rate (Cec¸en et al., 2010; Klapwuk et al., 1974; Ong et al.,2005).
Generally, trace elements of metals like iron, copper, manganese, zinc, chromium and selenium play unique as well as critical role since they are essential for our body metabolism and biochemical processes. These metals will form metalloprotein upon binding to protein and play their unique roles in enzyme reactions. The functions include as cofactor, stabilizer of protein's structure as well as facilitate in reactions of enzyme-substrate complexes to form end products (Fraga, 2005). Both deficiencies and excessive consumption of these metals might exhibit undesirable effects for health. As heavy metals exceed from the minutes amount of requirement, body tissues will be accumulated with these elements and the buildup concentration will exerts toxin properties. Heavy metals also can interfere with biochemical mechanism by preventing the proper function of a biochemical mechanism or by completely stopping its activity. The best example is enzymes, the cellular proteins that regulate many important chemical reactions. Heavy metals will react with enzymes and inhibit sulfhydryl (SH) enzymes systems.
A disturbance in enzymatic activity can seriously change the functioning of the organ or tissues. As examples, mercury and arsenic both bind to certain enzymes, thereby blocking their activity. Lead binds to the thiol chemical group in the enzymes and as a result, it will reduce the body's ability to synthesize enzymes necessary for respiration.
Table 1: Approximately order of toxicity of metals ion in descending order.
Highly toxic Decreasing toxicity ïƒ ïƒ ïƒ ïƒ
Cu Cd Au Ag Pt
Co K Ca Sr
Generally, a compound is considered toxic if it damages living organisms and it is defined in terms of the median lethal dose; LD50 is the amount of a compound, which causes death of 50% of the test animals (Rodericks, 1992). It is commonly expressed as the amount of the toxic compound per 100 grams or kilograms of the body weight of the test animal. While LC50 is the concentration of the toxic compound which kills 50% of the test animal in a given time.
Common symptoms of heavy metal poisoning include diarrhea, vomiting, pain in muscles and joints as well as chronic fatigue depending on how much concentration of metals being absorbed. The first step in diagnosing heavy metal poisoning is to screen by urine test, hair analysis or X-ray. However, hair test provides long-term record of metals exposure and the toxicity since hair acts as excretory tissue for heavy metals.
2.4 Detection of heavy metals
Heavy metals plays a major role and has been used in various field such as environmental studies, medicine and food industry but still shown its toxicity effects. Thus, rapid and sensitive measurements of heavy metals are required to make sure it is still under the permission limit.
2.4.1 Conventional method
Conventional methods or instrumental analysis such as atomic absorption spectroscopy, inductively coupled plasma optical emission spectrometry, inductively coupled plasma mass spectrometry and their combination with chromatographic techniques is in wide use. Electrochemical methods such as ion selective electrodes, polarography and other voltammetry are also widely used.
The advantages of these methods are the accuracy, specific and reproducible. Unfortunately, these methods have some disadvantages such as do not provide information about the toxicity of compounds to organisms, very expensive, need sophisticated instrumentation, skilled personnel, complicated sample pre-treatment and sometimes a long measuring period. Thus simple and fast procedures used as screening tests for industrial process water or foodstuffs to indicate the presence of toxic heavy metals are especially important (Han, 2001).
220.127.116.11 Atomic Absorption Spectrometry
Atomic absorption spectrometry (AAS) as shown in Figure 3 is considered as the most conventional method that has been used to detect heavy metals elements. It is an analytical method that measures the concentrations of elements. Atomic absorption is so responsive that it can measure down to parts per billion of a gram (µg dm-3) in a sample. Unfortunately, these methods have some disadvantages such as very expensive, need sophisticated instrumentation, skilled personnel, complicated and sometimes a long measuring period.
Figure 3: Atomic Absorption Spectrometry (AAS)
2.4.2 Bioassay for heavy metals
Bioindicator are referred as changes that occur at the organism, population and assemblage's levels when it has been exposed to toxic chemicals. Bioindicator using bacteria has been commercialized such as the PolytoxTM and the Microtox TM assays. Both of these two commercialized toxic detection kit are designed to detect a broad spectrum of toxic inorganic and organic pollutants.
The assay microorganisms in PolytoxTM are a blend of bacterial strains originally isolated from wastewater. The PolytoxTM kit as shown in Figure 4, specifically designed to assess the effect of toxic chemicals such as heavy metals on biological waste treatment and it is based on the reduction of the respiratory activity of the dehydrated cultures in the presence of toxicants. The PolytoxTM requires high cost and difficult to maintain. It is also needs sophisticated computer program to analysis data and highly trained technical personnel to run the tests, but it is not specific to certain group of chemicals.
Figure 4: PolytoxTM test kit
18.104.22.168 Microtox TM
Beckman Instruments Inc. developed the Microtox TM bioassay system as shown in Figure 5 as a screening instrument used for a variety of toxicity testing applications. The bioassay's strongest attribute is its convenience as a primary screening test for a wide spectrum of toxicants and its monitoring capability over time. The Microtox TM procedure can be used for testing either water (marine or fresh) or sediments.
Figure 5: Microtox TM 8 drug test cassette.
This assay requires a large budget and need a highly skilful person to run the test. It needs refrigerated water bath, a luminometer and sophisticated computer programmed to analyze the data (Botsford, 1996). And just as same as PolytoxTM test kit, it is not specific to certain group of chemicals.
22.214.171.124 MTT assay
Figure 6: Thiazolyl blue tetrazolium bromide
MTT assay is carried out using a tetrazolium dye 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction bacteria (Botsford, 1996). After reduction, the color of the respective MTT-formazan derivative is purple-blue. The reduction is inhibited by toxic compounds or chemicals thought to be toxic that damage living organism.
126.96.36.199 Enzymatic methods / bioassays
In recent years, the study of the effects of traces of metals ions on enzymatic activity has attracted considerable interest. Enzymatic methods can be used to determine the enzymes themselves, their substrates and effectors. Enzymatic determination of xenobiotics offers the simplest and rapid methods available for the testing of toxic xenobiotics. This bioassay does not require a skilled technician to operate, economic, faster and can detect synergistic effects of toxic compounds much better and more reliable than the classical methods.
The need for simpler, rapid and more importantly an economical system to monitor toxicity of heavy metals at the large scale level means that the simple enzyme inhibitive colorimetric assay of heavy metals using a simple spectrophotometer or color chart is urgently needed. So far bioassay using proteases such as trypsin, bromelain, papain, urease, phosphomolybdate reductase have been done (Shukor et al;2006,2007,2008). Future studies are predicted to come out with new source of protease which may develop bioassays with specific and sensitive detection towards heavy metals.