الفهرس 
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Abstract
Celiac disease is a chronic inflammatory disorder characterized by mucosal damage to the small intestine for children and adults. This disease is triggered by eating various foods containing gluten which found in wheat, barley and rye. Patient of celiac disease must not have any foods containing gluten. Gluten is a one of wheat proteins, which consists of glutenin and gliadin that causes celiac disease. from this point, the aim of this research was to evaluate the ability of different extracts of proteolytic enzymes from different biological sources to hydrolyze gluten and gliadin.
Protease enzymes were extracted from three sources:
1. Plant source is wheat grains germinated for 5 and 7 days, which contains cysteine protease enzymes.
2. Microbial source is Bacillus subtilis bacteria, which contains serine protease enzymes.
3. Insect source is larvae of red palm weevil aged 6 and 12 weeks, which contains serine protease enzymes.
Protease enzymes were purified by three means:
1. Ammonium sulfate at saturation of 40%, 60% & 80%.
2. Acetone at concentration of 40%, 60% & 80%.
3. Trichloroacetic acid at concentration of 0.2 M.
Evaluation of these purified extracts of protease enzymes from different sources on gluten and gliadin hydrolysis was monitored by the following determinations:
1- Determination of total protein in enzyme extracts.
2- Determination of total activity of protease enzymes.
3- Determination of specific activity of protease enzymes.
4- Determination of kinetic constants of protease enzymes.
5- Determination of precentage of gluten hydrolysis.
6- Determination of percentage of gliadin hydrolysis.
7- Determination of free amino acids after hydrolysis of gluten and gliadin.
The obtained results can be summarized as follows:
(1) Total activity and specific activity of protease enzymes:
The results showed that purification of protease enzymes extracted from wheat grains germinated for 5 and 7 days by trichloroacetic acid (TCA) at concentration of 0.2 molar give the highest total activity and specific activity and the lowest total protein content in comparison with purification by ammonium sulfate or acetone. Therefore, TCA was considered the best agent to purify protease enzymes extracted from germinated wheat grains. It is also noticed that the total activity and specific activity of protease enzymes extracted from wheat grains germinated for 7 days were higher than those for 5 days.
The results reveal that purification of protease enzymes extracted from Bacillus subtilis and larvae of palm weevil (age 6 and 12 weeks) by acetone 80% gave the highest total activity and specific activity and the lowest total protein content in comparison with purification by ammonium sulfate or trichloroacetic acid. It can be noticed that the total activity and specific activity of protease enzymes extracted from larvae of palm weevil aged 12 weeks were higher than those aged 6 weeks.
The comparison among three sources of protease enzymes, indicate that protease enzymes extracted from larvae of palm weevil aged 12 weeks and purified by acetone (80%) exhibited the highest specific activity followed by protease enzymes extracted from wheat grains germinated for 7 days and purified by TCA (0.2M), and then protease enzymes extracted from Bacillus subtilis and purified by acetone (80%). It is important to note that the total activity and specific activity of protease enzymes extracted from different sources were determined using casein as substrate.
(2) Kinetics of protease enzymes:
The results indicate that the constants, Km (Michaelis constant) and Vmax (maximum reaction velocity) of protease enzymes extracted from wheat grains germinated for 7 days were 0.607 mg/ml and 65.359 mg/min, respectively. It was found that these constants of protease enzymes extracted from Bacillus subtilis were 0.610 mg/ml and 105.263 mg/min, respectively. These constants of protease enzymes extracted from larvae of palm weevil aged 12 weeks were 0.129 mg/ml and 161.29 mg/min, respectively. The kinetics and constants of protease enzymes extracted from different sources demonstrate that the best source of protease enzymes was larvae of red palm weevil which exhibited the highest enzyme efficiency as indicated by the highest Vmax and the lowest Km. This was followed by the efficiency of protease enzymes extracted from Bacillus subtilis, and then germinated wheat grains.
These results were found to relatively varied with the previous determinations of total activity and specific activity of protease enzymes extracted from different sources which arranged in the order (larvae of palm weevil > germinated wheat grains > Bacillus subtilis). This may be due to using of bovine serum albumin (BSA) instead of casein as substrate in the determination of protease kinetics and constants.
(3) Hydrolysis of gluten and gliadin:
The aim of this study was to reach the maximum percentage of gluten and gliadin hydrolysis. To achieve this aim, several means are used in this study as follows: (1) using of protease enzymes from three different sources (plant, microbial and insect enzyme sources) (2) purification of protease enzyme extracts with three different precipitating agents (3) using of more than one of enzyme concentration (1 & 2 mg/ml) to hydrolyze gluten and gliadin (4) using of binary and tertiary mixtures of enzyme extracts from different sources to hydrolyze gluten and gliadin.
a) Gluten hydrolysis:
The data indicated that protease enzymes extracted from larvae of palm weevil give the highest percentage of gluten hydrolysis (32%) with enzyme concentration of 1 mg/ml during two hours of hydrolysis time. Other enzyme sources, Bacillus subtilis and germinated wheat grains gave lower percentages of gluten hydrolysis which accounted by 16 and 18% respectively. It can be noticed that increasing of enzyme concentration from 1 to 2 mg/ml exhibited pronounced increasing of gluten hydrolysis in case of protease enzymes extracted from germinated wheat grains (27% instead of 18%), slight increasing of gluten hydrolysis in case of protease enzymes extracted from Bacillus subtilis (19% instead of 16%), and no increasing of gluten hydrolysis in case of protease enzymes extracted from larvae of palm weevil (32%). Using of the tertiary mixture of protease enzymes from three different sources increased the percentage of gluten hydrolysis from 32% to 39 and 42% with enzyme concentration of 1 and 2 mg/ml respectively.
Comparatively, the percentages of gluten hydrolysis obtained by drug of protease enzymes (Digestin) were 35 and 42% with enzyme concentration of 1 and 2 mg/ml respectively. Another drug of protease enzymes (Polyzyme) gave the highest percentages of gluten hydrolysis (44 and 52%) with enzyme concentration of 1 and 2 mg/ml respectively. It means that the tertiary mixture of protease enzymes from three different sources gave similar percentages of gluten hydrolysis compared with Digestin drug and relatively lower percentages of gluten hydrolysis compared with Polyzyme drug.
b) Gliadin hydrolysis:
Protease enzymes extracted from three different sources were also examined to hydrolyze gliadin which was extracted from gluten because the gliadin fraction was reported to be responsible for celiac disease. In more details, the main responsible for celiac disease in gliadin was a polypeptide which consists of 33 amino acids with known sequence rich in proline and glutamine. These amino acids were reported to be responsible for resistance of this polypeptide for digestion by protease enzymes such as pepsin, trypsin and chemotrypsin. This polypeptide was reported to be resistant for digestion by protease enzymes not only in patients of celiac disease but also in all peoples. In healthy peoples this polypeptide does not permeate into their intact intestine and excrete in an undigested form. In patients of celiac disease this polypeptide does permeate into their damaged intestine and react with immunology system causing disease symptoms.
The data demonstrated that protease enzymes extracted from larvae of palm weevil give the highest percentage of gliadin hydrolysis (19%) at enzyme concentration of 1 mg/ml during two hours of hydrolysis time. Other enzyme sources, Bacillus subtilis and germinated wheat grains gave lower percentages of gliadin hydrolysis which accounted by 9 and 10% respectively. It is also noticed that increasing of enzyme concentration from 1 to 2 mg/ml exhibited marked increasing of gliadin hydrolysis in case of protease enzymes extracted from germinated wheat grains (16.8% instead of 10%) and slight increasing of gliadin hydrolysis in case of protease enzymes extracted from Bacillus subtilis (11.5% instead of 9%) and larvae of palm weevil (20% instead of 19%). Using of tertiary mixture of protease enzymes from three different sources increased the percentage of gliadin hydrolysis from 20% to 21 and 44% at enzyme concentrations of 1 and 2 mg/ml respectively.
Comparatively, the percentages of gliadin hydrolysis obtained by drug of protease enzymes (Digestin) were 16 and 43.6% at enzyme concentrations of 1 and 2 mg/ml respectively. Another drug of protease enzymes (Polyzyme) gave the highest percentages of gliadin hydrolysis (44 and 54.8%) at enzyme concentrations of 1 and 2 mg/ml respectively. It means that the tertiary mixture of protease enzymes from three different sources gave similar percentages of gliadin hydrolysis compared with Digestin drug and relatively lower percentages of gliadin hydrolysis compared with Polyzyme drug.
(4) Free amino acids after hydrolysis of gluten and gliadin:
The data illustrated that using of protein determination methods such as Bradford method to determine the efficiency of protease enzymes for degradation of gluten and gliadin were better than using of determination methods of total free amino acids such as Ninhydrin method for this purpose. This was mainly due to that hydrolysis of gluten and gliadin by protease enzymes produces only little amounts of free amino acids because most of these enzymes were reported to be endopeptidases. It means that a lot of breakdown of peptide ponds by action of protease enzymes does not produce free amino acids and consequently can not be measured by Ninhydrin method. Therefore, Ninhydrin method does not show the true efficiency of protease enzymes for protein degradation in contrary with Bradford method. It means that Ninhydrin method was found to be not suitable for measurement the efficiency of protease enzymes for degradation of gluten and gliadin.
The data demonstrated that protease enzymes extracted from larvae of palm weevil give the highest concentration of free amino acids produced by gluten hydrolysis (0.021 mg/ml) at enzyme concentration of 1 mg/ml during two hours of hydrolysis time. Other enzyme sources, Bacillus subtilis and germinated wheat grains gave lower concentrations of free amino acids which accounted by 0.005 and 0.006 mg/ml respectively. It can be noticed that increasing of enzyme concentration from 1 to 2 mg/ml does not exhibited any increase of free amino acids concentrations with all enzyme extracts. Using of the tertiary mixture of protease enzymes from three different sources increased the concentrations of free amino acids produced by gluten hydrolysis to reach 0.025 and 0.079 mg/ml at enzyme concentrations of 1 and 2 mg/ml respectively. The results also show that the concentrations of free amino acids produced by hydrolysis of gluten were 0.022 and 0.026 mg/ml by using of Digestin drug at enzyme concentrations of 1 and 2 mg/ml respectively. Using of Polyzyme drug for gluten hydrolysis gave free amino acids with concentrations of 0.024 and 0.029 mg/ml at enzyme concentrations of 1 and 2 mg/ml respectively.
The data also demonstrated that protease enzymes extracted from larvae of palm weevil give the highest concentration of free amino acids produced by gliadin hydrolysis (0.022 mg/ml) with enzyme concentration of 1 mg/ml during two hours of hydrolysis time. Other enzyme sources, Bacillus subtilis and germinated wheat grains gave comparable concentrations of free amino acids which accounted by 0.018 and 0.020 mg/ml respectively. It could be noticed that increasing of enzyme concentration from 1 to 2 mg/ml exhibit a slight increase only of free amino acids concentrations in case of Bacillus subtilis and larvae of palm weevil but an insignificant increase occurs in case of germinated wheat grains. Using of the tertiary mixture of protease enzymes from three different sources increased the concentrations of free amino acids produced by gliadin hydrolysis to reach 0.024 and 0.034 mg/ml at enzyme concentrations of 1 and 2 mg/ml respectively. The results also show that the concentrations of free amino acids produced by hydrolysis of gliadin were 0.025 and 0.031 mg/ml by using of Digestin drug with enzyme concentration of 1 and 2 mg/ml respectively. Using of Polyzyme drug for gliadin hydrolysis gave free amino acids with concentrations of 0.029 and 0.039 mg/ml at enzyme concentrations of 1 and 2 mg/ml respectively.
According to data of this study, tertiary mixture of protease enzymes extracted from wheat grains germinated for 7 days, Bacillus subtilis bacteria and larvae of palm weevil can be used as drug to overcome celiac disease or gluten sensitivity. This is mainly due to this mixture exhibited high efficiency for gluten and gliadin hydrolysis compared to drugs of protease enzymes such as Digestin and Polyzyme. These findings were proven through (1) determination of residual protein after hydrolysis of gluten and gliadin by action of different enzyme extracts by using of Bradford method, and (2) determination of free amino acids produced by hydrolysis of gluten and gliadin by using of Ninhydrin method.