The most abundant organic carbon source on Earth is cellulosic materials. Its main resources are ... more The most abundant organic carbon source on Earth is cellulosic materials. Its main resources are crop straws which are not commonly used and produce environmental pollution. These resources can be a site of biological hydrolysis to primary sugars by cellulase enzymes, in which avicelase is the most efficient enzyme in the cellulase family. This work aimed to clone the avicelase gene, transfer it to E. coli, optimize its expression, saccharify rice straw to its primary sugars, and ferment it to bioethanol. The avicelase gene was cloned from the Bacillus subtilis strain and cloned into two E. coli (i.e., DH5α and Bl21) strains. The optimized avicelase activity was described by testing the effect of different media and growth conditions including different carbon and nitrogen sources, as well as pHs and shaking or static conditions. Avicelase enzyme was extracted and used to saccharify rice straw. The obtained glucose was subjected to fermentation by Saccharomyces cerevisiae F.307 unde...
Susceptibility of Bacillus thuringiensis spores and toxin to UV-C (254nm) of the solar spectrum r... more Susceptibility of Bacillus thuringiensis spores and toxin to UV-C (254nm) of the solar spectrum reaching earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the Bt israeiensis (1977 &HD522) were more resistant to UV-C radiation than spores of at tenebrionfs (tt4) and the isolated strain (66). Maximum molluscicidal activities were obtained following successive rounds of exposing the 2 strains; tt4 and 66 to UV-C. The mutants obtained designated as 6615c & tt4/2a showed 100% mortality of Biomphalarla a/exandrina snails, while their parental strains showed 60% & 80% of mortality, respectively. The highly toxic mutants derived from at. (66 & tt4) appeared to produce number of crystals more than that of their parental strains. The protein pattems at these mutants indicated that they produced polypeptide of 65 KDa or its derivatives that caused high toxicity of these mutants. ,.. ~ DNA profile of some mutants was established by using polymerase chain reaction (PCR). 111e PCR screen can identify mutants with altered electrophoretic panerns containing novel genes.
Accumulation of feathers leads to environmental pollution and featherprotein wastage. Alkali hydr... more Accumulation of feathers leads to environmental pollution and featherprotein wastage. Alkali hydrolysis and cooking under steam pressure are the traditional ways to degrade feathers. They have many disadvantages such as destroying the amino acids; consume large amounts of energy and decrease protein quality and digestibility (Riffel and Brandelli, 2006). The enzymatic hydrolysis of feather may be a viable alternative to steam pressure cooking (Grazziotin et al., 2006).
Background and objectives The aim of this paper was to focus on improving production level of l-a... more Background and objectives The aim of this paper was to focus on improving production level of l-asparaginase by developing recombinant strains. Materials and methods Asp gene was cloned into the shuttle vector pNW33N, and the recombinant plasmid was used to transform Bacillus subtilis protoplast. Asp gene was expressed into both Escherichia coli and B. subtilis. Enzyme activity of the recombinant strains was measured as compared with wild-type strains. Results Asp gene was successfully subcloned into the recombinant plasmid named S-ASP-NRC-27. The gene was expressed efficiently in both host strains: E. coli and B. subtilis. The enzyme activity of the transformants was increased up to threefold under control of Lac Z promoter. Conclusion From the previous results, the shuttle vector pNW33N seemed to be a very useful plasmid as a cloning vector in a wide variety of the genus Bacillus. Both of Asp gene amplification and the control of Lac Z promoter had direct effects on producing the super Asp-expression strains.
Journal of Genetic Engineering and Biotechnology, 2018
The aim of this study is to construct a new recombinant strain able to degrade cellulose efficien... more The aim of this study is to construct a new recombinant strain able to degrade cellulose efficiently. The endo-b-1, 3-1, 4 glucanase (bgls) gene was cloned from Bacillus subtilis BTN7A strain by using PCR technique. The specific primers of bgls gene were deduced. Optimization of PCR mixture and program were identified. The nucleotide sequence of bgls was placed in the public domain (GenBank accession number KM009051.1). The obtained bgls DNA was cloned with pGEM Ò-T Easy Vector. The recombinant plasmid designated as Bgls-NRC-1 was transformed into E. coli DH5a. The successful cloning of the bgls gene was tested either by PCR or by evaluating its expression in its new bacterial host. The bgls gene was expressed efficiently in E. coli and the enzyme activity of the transformant was compared to the enzyme activity of the donor bacterial strain. The new constructs produce much higher enzyme yields than the donor bacterial strain, they produce about 29% and about 57% higher cellulase specific activity at 37°C and 55°C respectively. Optimization of cellulolytic activity of the new recombinant strain were described. The effect of minimal medium supplemented with CMC or cellulose, or complete medium (LB) on bgls expression were tested, the order of cellulase activity production was CMC27.2 > cellulose 21.9 > LB 19.8 U/mg protein, respectively at 24 h. CMC was proved to be the best medium for cellulase production. Results also showed that double the initial inoculum resulted in more cellulase activities in all media.
Journal of Genetic Engineering and Biotechnology, 2017
Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymet... more Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymethylcellulose) agar medium and screened by iodine method. Cellulase activity of the isolated bacteria was determined by DNS (dinitrosalicylic) acid method. The highly cellulolytic isolates (BTN7A, BTN7B, BMS4 and SA5) were identified on the basis of Gram staining, morphological cultural characteristics, and biochemical tests. They were also identified with 16S rDNA analysis. The phylogenetic analysis of their 16S rDNA sequence data showed that BTN7B has 99% similarity with Anoxybacillus flavithermus, BMS4 has 99% similarity with Bacillus megaterium, SA5 has 99% homology with Bacillus amyloliquefaciens and BTN7A was 99% similar with Bacillus subtilis. Cellulase production by these strains was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, different volumes of media, aeration rate and carbon source. The cellulase specific activity was calculated in each case. In conclusion four highly cellulolytic bacterial strains were isolated and identified and the optimum conditions for each one for cellulase production were determined. These strains could be used for converting plant waste to more useful compounds.
L-asparaginase has been used as a mainstay of all treatment of acute lymphoblastic leukemia. This... more L-asparaginase has been used as a mainstay of all treatment of acute lymphoblastic leukemia. This work amid to improve L-asparaginase production by constructing recombinant overproducing L-asparaginase strains through protoplast fusion technique between two highly L-asparaginase-producer local isolates, i.e., Bacillus subtilis and B. cereus. B. subtilis was sensitive to rifampcin (Rifs) and could utilize L-asparagine as a sole source of nitrogen while Bacillus cereus was resistant to (Rifr), does not grow on minimal medium and can not utilize L-asparagine. Protoplast was induced by treating bacterial cells with 1 mg/ml-1 lysozyme for three hours in SMM buffer. Protoplast regeneration was successfully obtained on sodium-succinate medium where protoplast regeneration rates were 39.8 and 25.6% for Bacillus subtilis and B. cereus, respectively. Protoplast fusion was performed between the two parental protoplasts in the presence of 40% PEG 6000. Fusants were obtained at high frequency, 17.7 x 10-2 on minimal M9 medium supplemented with rifampcin. Among forty five fusants, 18 showed significant higher L-asparaginase activity, they produced approximately 2.5fold more L-asparaginase. Optimization of physiological and fermentation factors for L-asparaginase overproducers hybrid strains were described. The obtained results indicated that protoplast fusion technique has a great potential for strain improvement particularly those used in industrial application.
Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to exam... more Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to examine the changes in alkaline protease gene expression following UV irradiation. Induction of mutation in Bacillus mutant strains was carried out by 0, 5, 10, 15 and 20 min. exposure times of UV irradiation and different distances between the treated bacterial cultures and UV source. Results revealed that alkaline protease activity assayed under submerged culture conditions was more accurate than the relative growth production (C/G) method, because there was no proportional correlation between zone diameter and the ability to produce the enzyme in submerged cultures. No enzyme activity was scored with B. pumilus mutants, while the activity was pronounce, in case of B. alvei mutants. Mutants No.15, 55 and 34 were the most efficient in enzyme production under submerged conditions being 68.8, 81.1 and 99 U/ml, respectively. Their alkaline protease activities were 2.6, 3.02 and 3.7 folds than those of the original strains. The optimal enzyme production was achieved after 48 hr at air: medium ratio of 39: 1. Results also proved that the inoculum size in all tested mutant ranges had no significant effect on the enzyme production .The supplementation with glucose to growth medium gave the highest level of enzyme productivity, while lactose showed the lowest. The addition of arabinose and xylose completely inhibited the enzyme production by both tested strains, while incorporation into the culture medium of sucrose and maltose failed to produce the enzyme in B. alvei, but in mutant No.8, they enhanced high levels of productivity.
The most abundant organic carbon source on Earth is cellulosic materials. Its main resources are ... more The most abundant organic carbon source on Earth is cellulosic materials. Its main resources are crop straws which are not commonly used and produce environmental pollution. These resources can be a site of biological hydrolysis to primary sugars by cellulase enzymes, in which avicelase is the most efficient enzyme in the cellulase family. This work aimed to clone the avicelase gene, transfer it to E. coli, optimize its expression, saccharify rice straw to its primary sugars, and ferment it to bioethanol. The avicelase gene was cloned from the Bacillus subtilis strain and cloned into two E. coli (i.e., DH5α and Bl21) strains. The optimized avicelase activity was described by testing the effect of different media and growth conditions including different carbon and nitrogen sources, as well as pHs and shaking or static conditions. Avicelase enzyme was extracted and used to saccharify rice straw. The obtained glucose was subjected to fermentation by Saccharomyces cerevisiae F.307 unde...
Susceptibility of Bacillus thuringiensis spores and toxin to UV-C (254nm) of the solar spectrum r... more Susceptibility of Bacillus thuringiensis spores and toxin to UV-C (254nm) of the solar spectrum reaching earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the Bt israeiensis (1977 &HD522) were more resistant to UV-C radiation than spores of at tenebrionfs (tt4) and the isolated strain (66). Maximum molluscicidal activities were obtained following successive rounds of exposing the 2 strains; tt4 and 66 to UV-C. The mutants obtained designated as 6615c & tt4/2a showed 100% mortality of Biomphalarla a/exandrina snails, while their parental strains showed 60% & 80% of mortality, respectively. The highly toxic mutants derived from at. (66 & tt4) appeared to produce number of crystals more than that of their parental strains. The protein pattems at these mutants indicated that they produced polypeptide of 65 KDa or its derivatives that caused high toxicity of these mutants. ,.. ~ DNA profile of some mutants was established by using polymerase chain reaction (PCR). 111e PCR screen can identify mutants with altered electrophoretic panerns containing novel genes.
Accumulation of feathers leads to environmental pollution and featherprotein wastage. Alkali hydr... more Accumulation of feathers leads to environmental pollution and featherprotein wastage. Alkali hydrolysis and cooking under steam pressure are the traditional ways to degrade feathers. They have many disadvantages such as destroying the amino acids; consume large amounts of energy and decrease protein quality and digestibility (Riffel and Brandelli, 2006). The enzymatic hydrolysis of feather may be a viable alternative to steam pressure cooking (Grazziotin et al., 2006).
Background and objectives The aim of this paper was to focus on improving production level of l-a... more Background and objectives The aim of this paper was to focus on improving production level of l-asparaginase by developing recombinant strains. Materials and methods Asp gene was cloned into the shuttle vector pNW33N, and the recombinant plasmid was used to transform Bacillus subtilis protoplast. Asp gene was expressed into both Escherichia coli and B. subtilis. Enzyme activity of the recombinant strains was measured as compared with wild-type strains. Results Asp gene was successfully subcloned into the recombinant plasmid named S-ASP-NRC-27. The gene was expressed efficiently in both host strains: E. coli and B. subtilis. The enzyme activity of the transformants was increased up to threefold under control of Lac Z promoter. Conclusion From the previous results, the shuttle vector pNW33N seemed to be a very useful plasmid as a cloning vector in a wide variety of the genus Bacillus. Both of Asp gene amplification and the control of Lac Z promoter had direct effects on producing the super Asp-expression strains.
Journal of Genetic Engineering and Biotechnology, 2018
The aim of this study is to construct a new recombinant strain able to degrade cellulose efficien... more The aim of this study is to construct a new recombinant strain able to degrade cellulose efficiently. The endo-b-1, 3-1, 4 glucanase (bgls) gene was cloned from Bacillus subtilis BTN7A strain by using PCR technique. The specific primers of bgls gene were deduced. Optimization of PCR mixture and program were identified. The nucleotide sequence of bgls was placed in the public domain (GenBank accession number KM009051.1). The obtained bgls DNA was cloned with pGEM Ò-T Easy Vector. The recombinant plasmid designated as Bgls-NRC-1 was transformed into E. coli DH5a. The successful cloning of the bgls gene was tested either by PCR or by evaluating its expression in its new bacterial host. The bgls gene was expressed efficiently in E. coli and the enzyme activity of the transformant was compared to the enzyme activity of the donor bacterial strain. The new constructs produce much higher enzyme yields than the donor bacterial strain, they produce about 29% and about 57% higher cellulase specific activity at 37°C and 55°C respectively. Optimization of cellulolytic activity of the new recombinant strain were described. The effect of minimal medium supplemented with CMC or cellulose, or complete medium (LB) on bgls expression were tested, the order of cellulase activity production was CMC27.2 > cellulose 21.9 > LB 19.8 U/mg protein, respectively at 24 h. CMC was proved to be the best medium for cellulase production. Results also showed that double the initial inoculum resulted in more cellulase activities in all media.
Journal of Genetic Engineering and Biotechnology, 2017
Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymet... more Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymethylcellulose) agar medium and screened by iodine method. Cellulase activity of the isolated bacteria was determined by DNS (dinitrosalicylic) acid method. The highly cellulolytic isolates (BTN7A, BTN7B, BMS4 and SA5) were identified on the basis of Gram staining, morphological cultural characteristics, and biochemical tests. They were also identified with 16S rDNA analysis. The phylogenetic analysis of their 16S rDNA sequence data showed that BTN7B has 99% similarity with Anoxybacillus flavithermus, BMS4 has 99% similarity with Bacillus megaterium, SA5 has 99% homology with Bacillus amyloliquefaciens and BTN7A was 99% similar with Bacillus subtilis. Cellulase production by these strains was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, different volumes of media, aeration rate and carbon source. The cellulase specific activity was calculated in each case. In conclusion four highly cellulolytic bacterial strains were isolated and identified and the optimum conditions for each one for cellulase production were determined. These strains could be used for converting plant waste to more useful compounds.
L-asparaginase has been used as a mainstay of all treatment of acute lymphoblastic leukemia. This... more L-asparaginase has been used as a mainstay of all treatment of acute lymphoblastic leukemia. This work amid to improve L-asparaginase production by constructing recombinant overproducing L-asparaginase strains through protoplast fusion technique between two highly L-asparaginase-producer local isolates, i.e., Bacillus subtilis and B. cereus. B. subtilis was sensitive to rifampcin (Rifs) and could utilize L-asparagine as a sole source of nitrogen while Bacillus cereus was resistant to (Rifr), does not grow on minimal medium and can not utilize L-asparagine. Protoplast was induced by treating bacterial cells with 1 mg/ml-1 lysozyme for three hours in SMM buffer. Protoplast regeneration was successfully obtained on sodium-succinate medium where protoplast regeneration rates were 39.8 and 25.6% for Bacillus subtilis and B. cereus, respectively. Protoplast fusion was performed between the two parental protoplasts in the presence of 40% PEG 6000. Fusants were obtained at high frequency, 17.7 x 10-2 on minimal M9 medium supplemented with rifampcin. Among forty five fusants, 18 showed significant higher L-asparaginase activity, they produced approximately 2.5fold more L-asparaginase. Optimization of physiological and fermentation factors for L-asparaginase overproducers hybrid strains were described. The obtained results indicated that protoplast fusion technique has a great potential for strain improvement particularly those used in industrial application.
Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to exam... more Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to examine the changes in alkaline protease gene expression following UV irradiation. Induction of mutation in Bacillus mutant strains was carried out by 0, 5, 10, 15 and 20 min. exposure times of UV irradiation and different distances between the treated bacterial cultures and UV source. Results revealed that alkaline protease activity assayed under submerged culture conditions was more accurate than the relative growth production (C/G) method, because there was no proportional correlation between zone diameter and the ability to produce the enzyme in submerged cultures. No enzyme activity was scored with B. pumilus mutants, while the activity was pronounce, in case of B. alvei mutants. Mutants No.15, 55 and 34 were the most efficient in enzyme production under submerged conditions being 68.8, 81.1 and 99 U/ml, respectively. Their alkaline protease activities were 2.6, 3.02 and 3.7 folds than those of the original strains. The optimal enzyme production was achieved after 48 hr at air: medium ratio of 39: 1. Results also proved that the inoculum size in all tested mutant ranges had no significant effect on the enzyme production .The supplementation with glucose to growth medium gave the highest level of enzyme productivity, while lactose showed the lowest. The addition of arabinose and xylose completely inhibited the enzyme production by both tested strains, while incorporation into the culture medium of sucrose and maltose failed to produce the enzyme in B. alvei, but in mutant No.8, they enhanced high levels of productivity.
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