In the present investigation partially purified alkaline protease from Aspergillus sp. As#6 and As#7 strains were entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature and pH maxima of protease from As#6 strain showed no changes before and after immobilization and remained stable at 450C and pH 9, respectively. However km value was slightly shifted from 4.5mg/ml to 5 mg/ml. Proteases from As#7 strain showed shifting in pH optima to a more alkaline range (10.0) as compared with free enzyme (9.0). Optimum temperature for protease from As#7 strain showed changes after immobilization and shifted from 650C to 850C. However there was no significant effect on Km value but Vmax of immobilized protease from As#7 strain was also shifted from 200U/ml to 370U/ml. Immobilized protease from As#6 strain was reused for 3 cycles with 22% loss in its activity whereas immobilize protease from As#7 strain was reused for 3 cycles with 17% loss in its activity. Protease from As#7 strain has a higher affinity for the substrate and higher proteolysis activity than protease from As#6 strain. The present work concludes that Aspergillus As#7 strain may be a good source of industrial protease
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Impact of anthelmintic efficacy of Calotropis procera on tegumental enzymes o...iosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Bioconversion of Penicillin to CephalosporinIOSR Journals
Cephalosporins are known as 3rd generation broad spectrum Beta lactam antibiotics, which can also be produced synthetically. Commonly, chemical ring expansion followed by an enzymatic removal of the phenylacetyl side chain is commonly employed to convert penicillin G into 7-aminodeacetoxycephalosporanic acid, the precursor for the manufacture of semisynthetic cephalosporins. This process requires several steps, is expensive and highly polluting. Thus there is a need to device a simple biological route to replace the chemical process. A mutant of Streptomyces clavuligerus NP1 was reported to converts Penicillin G to Deacetoxycephalosporin G (DAOG;phenylacetyl-7-aminodeacetoxycephalosporanic acid) enzymatically[5,8] . This enzyme, deacetoxycephalosporin synthase has the potential for the large scale transformation of Penicillin G to deacetoxycephalosporin. The present work studies the conditions required for efficient transformation of Penicillin G to Deacetoxycephalosporin using the wild type strain Streptomyces clavuligerus . Detection of cephalosporin was carried out using various methods. Additionally succinic acid formation was also studied as it could be used as a commercially important by product of the transformation. Deacetoxycephalosporin synthase also extracted and partially purified and characterised.
Optimum Conditions for Alginaseby Bacilllus Circulans R Isolateiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Optimization of Cultural Parameters for Cellulase Enzyme Production from Fung...IOSR Journals
Cellulalytic fungi synthesize cellulose enzyme for biodegradation of cellulose. This depends on various condition which include the source f isolation. This study was designed to determine the optimum condition necessary for cellulose production by fungi. Cellulose activities at different temperatures, pH and nitrogen sources by Rhizopus oryzae Aspergillus niger; A. flams, P. expansum and A. oryzae in liquid medium was studied and cellulose enzyme assay carried out by dinitrosalicylic acid method. All the fungal isolates have their highest cellulose activity at 400c except Penicillium expansum whose highest value of 1.28mg/ml was obtained at 320c. Cellulase produced 6m was found to be highest in all the isolate at pH 4.0 exception P expansum which occur at pH 5.5 (1.21mg/ml). The highest value e1.45mg/ml was obtained in A niger. Highest cellulose activity for A. niger, A. oryzae & P. expansum occurred in peptone. The study shows the need to determine the best physiological condition that allow for the optimal cellulose activity of fungal isolate. This will enhance their enzyme production.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Impact of anthelmintic efficacy of Calotropis procera on tegumental enzymes o...iosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Bioconversion of Penicillin to CephalosporinIOSR Journals
Cephalosporins are known as 3rd generation broad spectrum Beta lactam antibiotics, which can also be produced synthetically. Commonly, chemical ring expansion followed by an enzymatic removal of the phenylacetyl side chain is commonly employed to convert penicillin G into 7-aminodeacetoxycephalosporanic acid, the precursor for the manufacture of semisynthetic cephalosporins. This process requires several steps, is expensive and highly polluting. Thus there is a need to device a simple biological route to replace the chemical process. A mutant of Streptomyces clavuligerus NP1 was reported to converts Penicillin G to Deacetoxycephalosporin G (DAOG;phenylacetyl-7-aminodeacetoxycephalosporanic acid) enzymatically[5,8] . This enzyme, deacetoxycephalosporin synthase has the potential for the large scale transformation of Penicillin G to deacetoxycephalosporin. The present work studies the conditions required for efficient transformation of Penicillin G to Deacetoxycephalosporin using the wild type strain Streptomyces clavuligerus . Detection of cephalosporin was carried out using various methods. Additionally succinic acid formation was also studied as it could be used as a commercially important by product of the transformation. Deacetoxycephalosporin synthase also extracted and partially purified and characterised.
Optimum Conditions for Alginaseby Bacilllus Circulans R Isolateiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Optimization of Cultural Parameters for Cellulase Enzyme Production from Fung...IOSR Journals
Cellulalytic fungi synthesize cellulose enzyme for biodegradation of cellulose. This depends on various condition which include the source f isolation. This study was designed to determine the optimum condition necessary for cellulose production by fungi. Cellulose activities at different temperatures, pH and nitrogen sources by Rhizopus oryzae Aspergillus niger; A. flams, P. expansum and A. oryzae in liquid medium was studied and cellulose enzyme assay carried out by dinitrosalicylic acid method. All the fungal isolates have their highest cellulose activity at 400c except Penicillium expansum whose highest value of 1.28mg/ml was obtained at 320c. Cellulase produced 6m was found to be highest in all the isolate at pH 4.0 exception P expansum which occur at pH 5.5 (1.21mg/ml). The highest value e1.45mg/ml was obtained in A niger. Highest cellulose activity for A. niger, A. oryzae & P. expansum occurred in peptone. The study shows the need to determine the best physiological condition that allow for the optimal cellulose activity of fungal isolate. This will enhance their enzyme production.
Optimization of process parameters for l asparaginase production by aspergill...eSAT Journals
Abstract L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. L-asparaginase belongs to an amidase group that hydrolyses the amide bond in L-asparagine to aspartic acid and ammonia. The clinical action of this enzyme as an anti-carcinogenic is attributed to the reduction of L-asparagine; tumour cells unable to synthesise this amino acid are selectively killed by L-asparagine deprivation. L-Asparaginase has its application in food industry also. It helps in reducing the content of acrylamide in baked food products by hydrolysing the L-asparagine. L-Asparaginase is majorly produced by microorganisms including bacteria, yeast and fungi. The potential of Aspergillus terreus MTCC 1782 using cauliflower stalk: corn ears (3.75: 1.25) as substrate under SSF is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Various fermentation parameters such as types of agro material, their ratios, carbon source, nitrogen source, inoculum level, moisture content, temperature, pH, fermentation time, metal salts, and L-asparagine concentration, which influence the rate of enzyme production under SSF, were optimized. The optimized production of L-asparaginase has been obtained at 35°C for 4 days with a pH of 9.0, along with 50% moisture content, and 20% inoculum volume as the optimized fermentation conditions. The optimization was done using a ‘one-factor-at-a-time’ approach. The highest yield was obtained with, sucrose (1%w/v), ammonium sulphate (1%w/v), NaCl (1%w/v), L-asparagine (1%w/w), added to the fermentation medium, as supplements. Use of cauliflower stalk along with corn ear as potential raw materials for enzyme production could be of great commercial significance. Keywords: L-asparaginase, chemotherapeutic agent, Aspergillus terreus, SSF, mixed substrate, optimization
Optimization of l asparaginase production by aspergillus terreus mtcc 1782 us...eSAT Journals
Abstract Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that bring about chemical reactions concerned with life. They are protein in nature, colloidal and thermolabile in character, and specific in their action. L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. The present work deals with production of extracellular L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour under solid state fermentation Process parameters like Incubation time(96 h), Temperature (300 C), Moisture content (70% v/w), pH of the medium(8.0), Inoculum Age (5 days), Inoculum volume (1 ml), carbon source (1.5% w/v glucose), nitrogen source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of 273.3 U/gds of maximum L-asparaginase activity after optimization. The observation made in this study hold great promise for scale up production of L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour as substrate under solid state fermentation. Index terms: L-asparaginase, Aspergillus terreus, Bajra seed flour, Solid state fermentation, Optimization
Assessing the Suitability of using Plant Latex as Immobilization Support for ...ijsrd.com
Horseradish peroxidase was immobilized onto latex from three different plants viz. Calotropis procera, Euphorbia royleana and Alstonia scholaris with 0.51 ± 0.01, 0.37 ± 0.01, 0.46 ± 0.01 mg/cm2 conjugation yield and 62.07 ± 0.85, 66.1 ± 0.85, 71.24 ± 0.80 % retention of specific activity respectively. The support, before and after addition of peroxidase was characterized using scanning electron microscopy (SEM) and Fourier transmission infra-red spectroscopy (FTIR). Optimum pH, optimum temperature and changes in kinetic parameters (Ea, Km and Vmax) for immobilized peroxidases were studied and found to differ from that of free peroxidase. Alstonia scholaris latex was most effective in stabilizing the structure of peroxidase during storage at 4°C, whereas thermal stability and reusability of peroxidase was better on Calotropis procera latex. Analytical use of Calotropis procera latex bound peroxidase for determination of phenolic content of fruit juices has also been demonstrated.
Purification optimization and characterization of protease from Bacillus va...Vaibhav Maurya
This presentation is a research work carried out by me in B.Tech 8 semester. and gives an idea about purification, optimization and characterization of protease from Bacillus Valismortis
Industrial enzymes in the synthesis of drugs/ intermediatesPHARMA IQ EDUCATION
Importance of the following enzymes for the synthesis of drugs
1. introduction
2. penicillin acylase
3. lipase
4. oxidoreductase
5. transaminase
6. protease
Conclusion
References
Thanks
Asparaginase is an important enzyme in Medicine & food industry. It catalyzes Asparagine to aspartate and Ammonia. The purpose of using asparaginase in foods is to reduce the levels of acrylamide that form in certain carbohydrate-rich foods during cooking.The rationale behind asparaginase is that it takes advantage of the fact that acute lymphoblastic leukemia cells and some other suspected tumor cells are unable to synthesize the non-essential amino acid asparagine, whereas normal cells are able to make their own asparagine.
ABSTRACT- In this present study, the biotransformation of phenol to L-tyrosine was studied with resting cells of
Citrobacter freundii MTCC 2424 containing high tyrosine phenol lyase activity. Different process parameters leading to
synthesis of L-tyrosine were optimized. The L-tyrosine formed from biotransformation reactions was detected and
quantified by HPLC technique. The maximum L-tyrosine conversion 69% (6.49g/l) was obtained with ammonium
chloride 0.25M, phenol 0.1M, and sodium pyruvate 0.2M in borate buffer (0.1M) of pH 8.5 at 35°C temperature for
45min of incubation. Higher phenol concentration was found to be inhibitory for biotransformation due to phenol
inactivation of catalyst.
Key-words- Citrobacter freundii MTCC 2424, L-tyrosine, Tyrosine phenol lyase, Biotransformation
Anticancer Activity of L-asparaginase Produced from Amycolatopsis japonicaAI Publications
The ability of L-asparaginase to inhibit the formation of cancer cells has aroused scientists' curiosity in biological realms. In cancer cells, L-asparaginase suppresses protein synthesis by hydrolyzing L-asparagine to L-aspartic acid and ammonia. As a result, it's a crucial therapeutic enzyme in the treatment of Acute Lymphoblastic Leukemia in combination with other drugs (ALL). This enzyme has recently been discovered to be useful in a number of scientific fields, including clinical research, pharmacology, and the food business. Purification, characterization, and assessment of the cytotoxic effect of Amycolatopsis japonica L-asparaginase were the goals of this study. Amycolatopsis japonica was isolated from the plant rhizosphere and L-asparaginase was recovered. With a molecular weight of 37.5 KDa, partially purified L-asparaginase from A. japonica had a total activity of 1968.98 U with 26.696 mg total protein and a specific activity of 73.75 U/mg, 6.42 purification fold, and 42.86 percent recovery yield. In the presence of EDTA, Mg2+, pH8, 45oC, and 0.13 mM L-asparagine, L-asparaginase from A. japonica demonstrated good activity and stability, with Km and Vmax values of 0.13 mM L-asparagine and 0.43U/ mL, respectively. The cytotoxicity of L- asparaginase from A.japonica against a colon cancer cell line was high; with an IC50 value of 36 L. Amycolatopsis japonica could be a source of L-asparaginase, which could be a new target for cancer cells.
ABSTRACT- Microbial source of amylase is preferred to other sources because of its plasticity, vast availability, higher yield and
thermostability even at elevated temperatures.Various physical and chemical factors have been known to affect the production of α-
amylase such as temperature, pH, period of incubation, carbon sources acting as inducers, surfactants, nitrogen sources, phosphate,
different metal ions, moisture. Interactions of these parameters are reported to have a significant influence on the production of
the enzyme.Study was mainly aimed to isolate a bacterium capable of hydrolyzing a starch source and to check effect of different physiological
parameters on amylase enzyme activity. To conduct this research, study was mainly focused on three objectives i.e. 1st Screening
and morphological characterization of the isolated bacteria. 2nd Characterization of amylase production by selected isolates. 3rd
Time course of Enzyme production and Partial purification with Ammonium Sulphate saturation.Amylases of isolate-6 and isolate-9
were concentrated by ammonium sulfate precipitation which can be used as partially purified enzyme for further study. Isolate-6 and
Isolate-9 showed the activity 0.34 and 0.28 units/ml/min respectively.Enzyme derived from isolate-6 and isolate-9 was stable at different
physiological conditions. So, it is useful in fermentation industry and in pharmaceuticals.
Key words- Amylase, Starch hydrolyzing bacteria, fermentation and pharmaceutical industries
Optimization of process parameters for l asparaginase production by aspergill...eSAT Journals
Abstract L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. L-asparaginase belongs to an amidase group that hydrolyses the amide bond in L-asparagine to aspartic acid and ammonia. The clinical action of this enzyme as an anti-carcinogenic is attributed to the reduction of L-asparagine; tumour cells unable to synthesise this amino acid are selectively killed by L-asparagine deprivation. L-Asparaginase has its application in food industry also. It helps in reducing the content of acrylamide in baked food products by hydrolysing the L-asparagine. L-Asparaginase is majorly produced by microorganisms including bacteria, yeast and fungi. The potential of Aspergillus terreus MTCC 1782 using cauliflower stalk: corn ears (3.75: 1.25) as substrate under SSF is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Various fermentation parameters such as types of agro material, their ratios, carbon source, nitrogen source, inoculum level, moisture content, temperature, pH, fermentation time, metal salts, and L-asparagine concentration, which influence the rate of enzyme production under SSF, were optimized. The optimized production of L-asparaginase has been obtained at 35°C for 4 days with a pH of 9.0, along with 50% moisture content, and 20% inoculum volume as the optimized fermentation conditions. The optimization was done using a ‘one-factor-at-a-time’ approach. The highest yield was obtained with, sucrose (1%w/v), ammonium sulphate (1%w/v), NaCl (1%w/v), L-asparagine (1%w/w), added to the fermentation medium, as supplements. Use of cauliflower stalk along with corn ear as potential raw materials for enzyme production could be of great commercial significance. Keywords: L-asparaginase, chemotherapeutic agent, Aspergillus terreus, SSF, mixed substrate, optimization
Optimization of l asparaginase production by aspergillus terreus mtcc 1782 us...eSAT Journals
Abstract Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that bring about chemical reactions concerned with life. They are protein in nature, colloidal and thermolabile in character, and specific in their action. L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. The present work deals with production of extracellular L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour under solid state fermentation Process parameters like Incubation time(96 h), Temperature (300 C), Moisture content (70% v/w), pH of the medium(8.0), Inoculum Age (5 days), Inoculum volume (1 ml), carbon source (1.5% w/v glucose), nitrogen source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of 273.3 U/gds of maximum L-asparaginase activity after optimization. The observation made in this study hold great promise for scale up production of L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour as substrate under solid state fermentation. Index terms: L-asparaginase, Aspergillus terreus, Bajra seed flour, Solid state fermentation, Optimization
Assessing the Suitability of using Plant Latex as Immobilization Support for ...ijsrd.com
Horseradish peroxidase was immobilized onto latex from three different plants viz. Calotropis procera, Euphorbia royleana and Alstonia scholaris with 0.51 ± 0.01, 0.37 ± 0.01, 0.46 ± 0.01 mg/cm2 conjugation yield and 62.07 ± 0.85, 66.1 ± 0.85, 71.24 ± 0.80 % retention of specific activity respectively. The support, before and after addition of peroxidase was characterized using scanning electron microscopy (SEM) and Fourier transmission infra-red spectroscopy (FTIR). Optimum pH, optimum temperature and changes in kinetic parameters (Ea, Km and Vmax) for immobilized peroxidases were studied and found to differ from that of free peroxidase. Alstonia scholaris latex was most effective in stabilizing the structure of peroxidase during storage at 4°C, whereas thermal stability and reusability of peroxidase was better on Calotropis procera latex. Analytical use of Calotropis procera latex bound peroxidase for determination of phenolic content of fruit juices has also been demonstrated.
Purification optimization and characterization of protease from Bacillus va...Vaibhav Maurya
This presentation is a research work carried out by me in B.Tech 8 semester. and gives an idea about purification, optimization and characterization of protease from Bacillus Valismortis
Industrial enzymes in the synthesis of drugs/ intermediatesPHARMA IQ EDUCATION
Importance of the following enzymes for the synthesis of drugs
1. introduction
2. penicillin acylase
3. lipase
4. oxidoreductase
5. transaminase
6. protease
Conclusion
References
Thanks
Asparaginase is an important enzyme in Medicine & food industry. It catalyzes Asparagine to aspartate and Ammonia. The purpose of using asparaginase in foods is to reduce the levels of acrylamide that form in certain carbohydrate-rich foods during cooking.The rationale behind asparaginase is that it takes advantage of the fact that acute lymphoblastic leukemia cells and some other suspected tumor cells are unable to synthesize the non-essential amino acid asparagine, whereas normal cells are able to make their own asparagine.
ABSTRACT- In this present study, the biotransformation of phenol to L-tyrosine was studied with resting cells of
Citrobacter freundii MTCC 2424 containing high tyrosine phenol lyase activity. Different process parameters leading to
synthesis of L-tyrosine were optimized. The L-tyrosine formed from biotransformation reactions was detected and
quantified by HPLC technique. The maximum L-tyrosine conversion 69% (6.49g/l) was obtained with ammonium
chloride 0.25M, phenol 0.1M, and sodium pyruvate 0.2M in borate buffer (0.1M) of pH 8.5 at 35°C temperature for
45min of incubation. Higher phenol concentration was found to be inhibitory for biotransformation due to phenol
inactivation of catalyst.
Key-words- Citrobacter freundii MTCC 2424, L-tyrosine, Tyrosine phenol lyase, Biotransformation
Anticancer Activity of L-asparaginase Produced from Amycolatopsis japonicaAI Publications
The ability of L-asparaginase to inhibit the formation of cancer cells has aroused scientists' curiosity in biological realms. In cancer cells, L-asparaginase suppresses protein synthesis by hydrolyzing L-asparagine to L-aspartic acid and ammonia. As a result, it's a crucial therapeutic enzyme in the treatment of Acute Lymphoblastic Leukemia in combination with other drugs (ALL). This enzyme has recently been discovered to be useful in a number of scientific fields, including clinical research, pharmacology, and the food business. Purification, characterization, and assessment of the cytotoxic effect of Amycolatopsis japonica L-asparaginase were the goals of this study. Amycolatopsis japonica was isolated from the plant rhizosphere and L-asparaginase was recovered. With a molecular weight of 37.5 KDa, partially purified L-asparaginase from A. japonica had a total activity of 1968.98 U with 26.696 mg total protein and a specific activity of 73.75 U/mg, 6.42 purification fold, and 42.86 percent recovery yield. In the presence of EDTA, Mg2+, pH8, 45oC, and 0.13 mM L-asparagine, L-asparaginase from A. japonica demonstrated good activity and stability, with Km and Vmax values of 0.13 mM L-asparagine and 0.43U/ mL, respectively. The cytotoxicity of L- asparaginase from A.japonica against a colon cancer cell line was high; with an IC50 value of 36 L. Amycolatopsis japonica could be a source of L-asparaginase, which could be a new target for cancer cells.
ABSTRACT- Microbial source of amylase is preferred to other sources because of its plasticity, vast availability, higher yield and
thermostability even at elevated temperatures.Various physical and chemical factors have been known to affect the production of α-
amylase such as temperature, pH, period of incubation, carbon sources acting as inducers, surfactants, nitrogen sources, phosphate,
different metal ions, moisture. Interactions of these parameters are reported to have a significant influence on the production of
the enzyme.Study was mainly aimed to isolate a bacterium capable of hydrolyzing a starch source and to check effect of different physiological
parameters on amylase enzyme activity. To conduct this research, study was mainly focused on three objectives i.e. 1st Screening
and morphological characterization of the isolated bacteria. 2nd Characterization of amylase production by selected isolates. 3rd
Time course of Enzyme production and Partial purification with Ammonium Sulphate saturation.Amylases of isolate-6 and isolate-9
were concentrated by ammonium sulfate precipitation which can be used as partially purified enzyme for further study. Isolate-6 and
Isolate-9 showed the activity 0.34 and 0.28 units/ml/min respectively.Enzyme derived from isolate-6 and isolate-9 was stable at different
physiological conditions. So, it is useful in fermentation industry and in pharmaceuticals.
Key words- Amylase, Starch hydrolyzing bacteria, fermentation and pharmaceutical industries
Proteases are protein-degrading enzymes that catalyses hydrolytic reaction in which protein molecules are degraded into peptides and amino acids. Thermostable alkaline proteases are of particular great interest for industrial application because they are stable and active at temperature above 60-70˚C. Thermophiles are found in wide array of environment such as mushroom compost material, nest, hay, wood chips, grains, soil, manure, coal mines etc. Alkaline proteases are most important industrial enzymes and they occupy about 60% of total enzyme market. From the soil samples, eight different fungal species were isolated through soil dilution plate method. In the present study, two fungi Aspergillus nidulans and Aspergillus glaucus from mushroom compost and two fungi Aspergillus terrus, and Aspergillus fumigates from cow manure, showing alkaline protease activity, were isolated. The zones of clearance were observed in Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species of fungi isolated from cow manure and mushroom compost. The best enzyme production was observed in Aspergillus terrus (1.005 ± 0.057 IU/mg protein) obtained from cow manure and the minimum enzyme activity was observed with Aspergillus glaucus (0.278 ± 0.026 IU/mg protein). However, more studies are required to assess the potential of Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species. Key-words- Alkaline protease, Thermophiles, Zone of clearance, Trichloroacetic acid
Effect of Different Physico-Chemical Parameters on Production ofAmylase by Ba...IOSR Journals
The present study is concerned with the production of amylase by Bacillus species strain. In this
study 12 bacterial strains were isolated and screened for their α-amylase activity. These strains were
maintained on nutrient agar medium. Fermentation for the production of amylase was carried out in the enzyme
production medium (EPM). All the 12 strains were tested for amylase production. On the basis of maximum
amylase activity strain no.1 was selected for further studies. Different starch concentrations, 0.75,1.00,1.25%,
pH labels 6.5,7.0,7.5,8.0, aeration (RPM), 100,120,140, temperatures 250C,280C,370C, and 400C and inoculums
level 0.5%,1.0%, 1.5% and 2.0% were studied
Lipase production and purification Likhith KLIKHITHK1
Lipase (tri acyl glycerol acyl hydrolase, EC 3.1.1.3) catalyzes the hydrolysis of the carboxyl ester bonds in tri acyl glycerols to produce di acyl glycerols, mono acyl glycerols, fatty acids and glycerol under aqueous conditions and the synthesis of esters in organic solvents.
Under the controlled conditions, lipases are able to catalyze a large number of reactions. Lipases of microbial origin are of considerable commercial importance, because of the high versatility and high stability, moreover, the advantage of being readily produced in high yields.
Many microbial lipases have been commercially available in free or immobilized form. Numerous species of bacteria (Bacillus, Pseudomonas, and Burkholderia), yeasts (Candida rugosa, Yarrowia lipolytica, and Candida antarctica) and molds (Aspergillus, Trichoderma viride) produce lipases with different enzymological properties and specificities but microbes are known to be more potent lipase producer.
Glutathione S-transferase enzymes (GSTs) play central roles in phase II detoxification of both xenobiotics and endogenous compounds in almost all living organisms. The enzyme was extracted and partially purified from wheat leaves through a procedure including ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures yielded a 7.14-fold purification with 71% recovery. Optimum activity conditions-pH, temperature and ionic strength-of the enzyme were determined. Its some kinetic properties such as Vmax, KM, and kcat were calculated for GSH and CDNB substrates. The kcat/KM values of the enzyme were 603.5 for GSH and 385.3 for CDNB. The native molecular weight of the enzyme was estimated to be 52 kDa based on its mobility in gel filtration column.
Isolation and purification of peroxidase from soyabeanPooja Walke
Peroxidase (EC. 1.11.1.7), an oxidoreductase, has iron porphyrin ring generally and catalyzes a redox reaction between H202 as an electron acceptor and many kinds of substrates by means of oxygen liberation from HzOz (Brill, 1996).
IJPCBS 2012, 2(1), 110-116 Kavya et al. ISSN: 2249-9504
110
INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES
Available online at www.ijpcbs.com
ISOLATION AND SCREENING OF STREPTOMYCES SP. FROM
CORINGA MANGROVE SOILS FOR ENZYME PRODUCTION AND
ANTIMICROBIAL ACTIVITY
M. Kavya Deepthi1*, M. Solomon Sudhakar1 and M. Nagalakshmi Devamma2 1Department of Biotechnology, Rajalakshmi Engineering College, Thandalam, 2Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pr Taadmesihln, aInddui,a I.n dia.
Immobilization of two endoglucanases from different sourcesIJEAB
Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Kinetic study of free and immobilized protease from Aspergillus sp.
1. IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 7, Issue 2 (Jul. – Aug. 2013), PP 86-96
www.iosrjournals.org
www.iosrjournals.org 86 | Page
Kinetic study of free and immobilized protease from Aspergillus
sp.
Namrata Sharma and ShwetaTripathi
Deptt. of Biochemistry. Mata Gujri Mahila Mahavidyalaya, Jabalpur (M.P.)
Abstract: In the present investigation partially purified alkaline protease from Aspergillus sp. As#6 and As#7
strains were entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature
and pH maxima of protease from As#6 strain showed no changes before and after immobilization and
remained stable at 450
C and pH 9, respectively. However km value was slightly shifted from 4.5mg/ml to 5
mg/ml. Proteases from As#7 strain showed shifting in pH optima to a more alkaline range (10.0) as compared
with free enzyme (9.0). Optimum temperature for protease from As#7 strain showed changes after
immobilization and shifted from 650
C to 850
C. However there was no significant effect on Km value but Vmax of
immobilized protease from As#7 strain was also shifted from 200U/ml to 370U/ml. Immobilized protease from
As#6 strain was reused for 3 cycles with 22% loss in its activity whereas immobilize protease from As#7 strain
was reused for 3 cycles with 17% loss in its activity. Protease from As#7 strain has a higher affinity for the
substrate and higher proteolysis activity than protease from As#6 strain. The present work concludes that
Aspergillus As#7 strain may be a good source of industrial protease.
Key Words: Alkaline protease, Aspergillus sp, Immobilized protease, Km, pH, Temperature, Vmax.
I. Introduction
The continuous requirement of proteases has made them industrially important enzyme. They account
for 65% of total world wide enzyme sale [1]. They have major applications in industrial process such as laundry
detergents, leather, silk, pharmaceutical, food and degradation of gelatin on X-ray films [1, 2, 3, 4]. They are
also used in bioremediation process [5].
Proteases are generally distributed in nature. But proteases of microbial origin possess industrial
potential due to their biochemical diversity. A variety of microorganisms such as bacteria, fungi, yeast and
actinomycetes are known to produce these enzymes [6]. It would be beneficial to use a fungal protease as
fungal expression systems are capable of producing larger quantities of enzymes than bacterial expression
system [7]. Proteases are produced by many species of fungi such as Aspergillus[8], Mucor [9], Fusarium [10],
Cephalosporium [11] and Rhizopus [12]. Filamentous fungi, such as Aspergillus have been the organism of
choice for large scale production of bulk industrial enzymes. [13].
For commercial application of enzyme it must be produced at low cost and should be reused, reproduce
result with consistent efficiency. Thus different methods have been used to reduce the cost and increase the
utilization of proteases, one of which is immobilization. Immobilized enzymes offer advantages which include
high productivity, automation, continuous processing, precise control of the extent of reaction, easy product
recovery and the enzyme does not contaminate the final product [14]. Additional benefits arise from
stabilization against harsh conditions which are deleterious to soluble enzyme preparations. The properties of
immobilized enzyme preparations are governed by the properties of both enzyme and the carrier material. The
specific interaction between the latter provides an immobilized enzyme with distinct chemical, biochemical,
mechanical and kinetic properties. Calcium alginate is the most widely used matrix for entrapment of enzyme.
Entrapment within insoluble calcium alginate is recognized as a rapid, nontoxic, inexpensive and versatile
method for immobilization of enzyme as well as cells [15].
The present study reports the kinetic study of extracellular free and immobilized protease from local
soil isolate of Aspergillus sp, AS#6 and AS#7 .These protease were partially purified and were immobilized in
calcium alginate beads. Then their kinetic properties were studied.
II. Materials And Methods
Micro organism and maintenance of culture -
The fungal strains used throughout this study were isolated from soil samples of Jabalpur area. These
isolates were identified as of Aspergillus sp, on the basis of their morphological and microscopic identification.
The Aspergillus Strains AS#6 and AS#7 were maintained on potato dextrose agar plates at 28 ±20
C. Spores for
inoculums were prepared from 5-7 days old cultures by sterile cork borer in Tween - 80 solution.
2. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 87 | Page
Growth Conditions -
The medium used for protease production by Aspergillus sp, was Yeast Extract Broth, composed of
Yeast Extract, 0.5%, KCl 2%, Peptone, 2%, Sucrose 2%, Casein, 1.0%, pH 7.5. YE broth was autoclaved at
1210
C for 15 mins. Broth was inoculated with two Aspergillus strains AS#6 and AS#7 and incubated in a rotary
shaker at 150 rpm for 96h at 28 ±20
C, in separate 250 ml Erlenmeyer flasks with working volume of 100 ml.
The cultures were centrifuged at 10,000 rpm for 10 min at 40
C to remove fungal mycelia and supernatants were
used as the crude enzyme solution.
Assay of protease activity -
Protease activity was measured by the method of Anson [16], using casein as a substrate. A control
lacking the enzyme was included in each assay.
One unit of protease hydrolyzed casein to produce color equivalent to 1.0μmol (181μg) of tyrosine per
minute at pH 7.5 at 370
C. The enzyme activity was expressed as U/ml.
Determination of Protein content -
The protein content of fraction, obtained after ammonium sulphate precipitation was determined by the
method of Lowry [17] using BSA as a substrate.
Ammonium Sulphate fractionation -
Solid ammonium sulphate was added to the crude extract to 0-50% saturation. The precipitate was
collected by centrifugation, dissolved in minimal volume of Tris-HCl buffer (pH 7.8) and desalted by using
prepacked desalting column.
Protease Kinetics -
1. Effect of different substrates on hydrolytic action of protease -
To study the effect of various substrates, 1 ml of the partially purified enzyme solution of the
Aspergillus strains were incubated with 5 ml of each of these substrate (casein, gelatin, BSA and Hemoglobin
0.65% w/v) for 10 min at 370
C, separately. After incubation the enzyme activities were determined.
2. Effect of initial enzyme concentration on casein hydrolysis.
For this, the reaction mixtures were prepared varying the enzyme concentration from 12U/ml to
110U/ml. 5 ml of substrate solutions was added to each of the above enzyme concentrations, then these were
incubated at 370
C for 10 min for protease obtained from both the strains AS#6 and AS#7. Then enzyme assay
was done under standard conditions.
3. Effect of initial substrate concentration on casein, hydrolysis
In this experiment reaction mixtures were designed varying the substrate concentration from 1mg/ml to
7 mg/ml to which 1ml of partially purified protease solution from the two Aspergillus strains AS#6 and AS#7
were added in two separate set of experiments. The reaction mixutres were then incubated at 370
C for 10 min
and then protease activity was determined. Kinetic parameters Km and Vmax were calculated from Line weaver
Burk Plots [8].
4. Effect of pH on casein hydrolysis
To evaluate casein hydrolysis at different pH (ranging from 5-10), the 1 ml of enzyme solution was
added to the 0.65% casein solution by adjusting the pH from 5-10. For pH 5 acetate buffer, pH 6-7 phosphate
buffer, pH 8-9 tris buffer, pH-10 glycine - NaOH buffer were used. Protease activity was determined under
standard assay conditions.
5. Effect of reaction temperature on casein hydrolysis -
Optimum temperature for activity of the protease was determined by carrying out the reactions at
temperatures ranging from 0O
C to 650
C. At each temperature the substrate was pre incubated at the required
temperature before the addition of the enzyme.
Immobilization method -
Entrapment -
The partially purified protease from AS#6 and AS#7 strains were immobilized in the calcium alginate
beads through entrapment by the method of Banerjee et al., [19]. Bovine serum albumin (5mg) was added to 5.0
ml of enzyme solution. To this 1.5% of sodium alginate was added and stirred gently. The entrapment was
3. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 88 | Page
carried out by dropping the mixture through a glass pipette into 50 ml of 2.0% (W/v) CaCl2 solution. The beads
so formed were left for 1h in calcium chloride solution and then stored in 0.1 M Tris HCl buffer, pH 9.0 at 40
C.
Calculation for protease from AS#6 strain [20]
Initial activity of the free enzyme = 297U/ml
Volume of enzyme solution = 5 ml
Weight of beads fromed after immobilization of enzyme solution = 2.5 gm
Enzyme solution enterapped in 0.5 g beads = 0.98 ml
Therefore enzyme enterapped = 199U/0.5g beads
Activity of immobilized enzyme obtained in 0.5 g beads = 192U/0.5g beads
Therefore total enzyme activity after entrapment = 96%
Protease from AS#7 strain
Initial activity of the free enzyme = 203.5U/ml
Volume of enzyme solution = 5 ml
Weight of beads formed after immobilization of enzyme solution = 1.63 gm
Enzyme solution entrapped in 0.5 g beads = 1.53 ml
Activity of immobilized enzyme obtained in 0.5 g beads = 324.36U/0.5g beads
Therefore enzyme entrapped = 228.25U/0.5g beads
Therefore total enzyme activity after entrapment = 76%
Kinetic properties of immobilized protease –
1. Effect of pH and temperature on the immobilized protease-
Effect of pH and temperature on the immobilized protease was determined under standard assay
conditions using casein as a substrate. Protease activity was studied in the pH range from 5-10 for immobilized
form of enzymes and then their activity was measured at various temperatures (00
C to 650
C). Per assay tube 5
beads of immobilized enzyme were added.
2. Effect of substrates concentration on immobilized protease -
The Km and Vmax value were determined for the hydrolysis of casein by immobilized protease. A
Lineweaver Burk plot was drawn between the inverse of different concentration of casein and reaction velocity
to determine the Michaelis constant Km and Vmax for immobilized protease.
III. Results and Discussion
Partial Purification -
A better understanding of the functions of enzymes could be determined by purification of enzyme
[21]. Partial purification of the enzyme by ammonium sulphate precipitation followed by desalting through
prepacked desalting column of dextrin, resulted in nearly 7.5 fold increase in the specific activity of the enzyme
(Table- 1) from AS#6 strain and 19.2 fold increase in the specific activity of the protease from AS#7 strain
(Table - 2).
Protease Kinetics (Free Form) -
Enzyme Kinetics is the most fundamental aspect of enzymology. For the commercial exploitation of
enzyme it is desirable to choose an enzyme, which will have the fastest reaction rate per unit amount of enzyme.
Reaction rate indicates the maximum effect for minimum amount of added catalyst [22].
Effect of initial substrate concentration on casein hydrolysis -
The assay substrate, casein, when used in increasing concentrations resulted in the saturation of the
enzyme (Fig -1). There was an increase in substrate hydrolysis upto 6 mg/ml in both the Aspergillus strains
AS#6 and AS#7 and thereafter there was saturation of the enzyme. From Michaelis-Menten equation it is seen
that the rate of enzyme reaction is directly proportional to the substrate concentration when the concentration of
substrate is low. But at higher magnitude the reaction rate is not influenced by substrate concentration [23].
From Lineweaver Burk Plot, the Km and Vmax of the reaction was found to be 4.5 mg/ml and 142.8
U/ml, respectively for protease obtained from AS#6 strain (Fig 3) whereas protease from AS#7 strain showed
Km and Vmax value 2.5 mg/ml and 200 U/ml, respectively (Fig : 4) Since protease from AS#7 strain has a
higher afinity for the substrate and highest proteolytic activity than protease from AS#6 strain therefore it
suggests that partially purified protease from AS#7 strain may have more industrial applications.
4. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 89 | Page
Effect of enzyme concentration on casein hydrolysis-
The study of effect of initial enzyme concentration in Fig:5,6 showed that at low concentration the rate
of hydrolysis increased to linearly. It was observed from figure that 89.3 U/ml and 69.5 U/ml enzyme
concentration was optimum for protease from AS#6 and AS#7 strains, respectively. This may be due to the
limitation of availability of substrate for the active sites of enzyme and for which further addition of the enzyme
could not enhance the rate of product formation [24].
Effect of different substrates on hydrolytic action of protease -
Among the various substrates studied, casein was found to be the best substrate for protease obtained
from both the strain AS#6 and AS#7 (Fig : 7, 8 ). When gelatin and BSA were used as the substrates, protease
from AS#7 strain showed no proteolysis activity. Protease obtained from AS#6 strain showed highest
proteolysis activity in casein followed by hemoglobin, gelatin and BSA.
Effect of pH on casein hydrolysis -
Enzymes, being proteinaceous in nature, have properties that are quite pH sensitive. pH can affect
activity by changing the charges on an amino acid residue which is functional in substrate binding or catalysis.
Partially purified protease from AS#6 and AS#7 strains both showed maximum activity at pH 9 (Fig 9,
10). A fall in the hydrolysis rate on either side of the optimum value is due to decrease in affinity of enzyme for
the substrate [23]. Similar observation was reported in a new strain of Aspergillus Oryzae AWT 20. The
optimum pH of the free enzyme of this strain was 9.0 [25].
Alkaline protease from Bacillus circulans also show activity over broad pH range (8-11) with
optimum pH at 9.0 [26].
Effect of reaction temperature on casein hydrolysis -
Partially purified protease from Aspergillus AS#6 strain showed increased rate of enzyme reaction with
increase in temperature up to 450
C but thereafter activity decreased whereas partially purified protease from
AS#7 strain showed optimum activity at 650
C (Fig : 11, 12). Increase in temperature above optimum level
affects important factors like protein denaturation, protein ionization state and solubility of species in solution
reducing enzyme activity [27]. The optimum temperature of protease obtained from AS#7 strain was slightly
higher than those from other fungal proteases such as Rhizopus oryzae [28] and Aspergillus niger 1 [29] which
exhibited optimum temperature at 600
C. Aspergillus niger NRRL 1785 exhibited optimum temperature at 500
C
[30] which is slightly higher than from protease of AS#6 strain which showed optimum activity at 450
C.
Immobilized protease Kinetics -
Effect of substrate concentration on activity of immobilized protease
Michaelis - Menten (Km) value of entrapped protease was determined by studying the hydrolysis of
different concentration of substrate (casein), ranging from 1mg/ml to 7 mg/ml. Line weaver Burk Plot was
drawn between the inverse of different concentrations of casein and reaction velocity to determine the constant
for immobilized protease. The Km and Vmax value for immobilize protease from AS#6 strain was found 5
mg/ml and 167 U/ml, respectively, (Fig :13). Protease from AS#7 strain showed 2.6 mg/ml Km and 370 U/ml
Vmax value, (Fig : 14) respectively for immobilized enzyme while Km and Vmax for free enzyme was 2.5
mg/ml and 200 U/ml/min, respectively. This increase in substrate concentration might be due to the inability of
high molecular weight substrate casein to diffuse rapidly into the Ca-alginate matrix [31]. AS#7 strain showed
85% increase in Vmax value of the immobilized enzyme as compared to free enzyme. This may be due to the
rate of diffusion of substrate to the enzyme may rise to a limiting value as the substrate concentration in the bulk
solution is increased. If this limiting value is reached after an immobilized enzyme is completely saturated with
substrate, then its apparent Vmax value will high than that in free solution [32].
Effect of temperature and pH on activity of immobilized protease -
Alginate entrapped enzyme was assayed at different temperature and pH ranging from 30-850
C and pH
5-10, respectively, The optimum temperature of entrapped enzyme from AS#6 and AS#7 strain was 450
C and
850
C respectively (Fig: 15, 16). In the present case for protease from AS#6 strain the optimum temperature
(450
C) and pH (9) (fig- 17) of free and immobilized enzyme remain the same. Consistently, Arya and Srivastava
[33] and Anwar et al, [34] reported that no change occurred in the optimum temperature and pH of CGtase and
protease, respectively, before and after entrapment in calcium alginate beads.
The optimum pH value of the immobilized protease form AS#7 strain shifted to pH 10 from 9.0 which
was the optimum pH of the free enzyme (Fig 10). This may be due to the anionic nature of the alginate support
used for immobilization and change in microenvironment of the immobilized enzyme. Similar observation
reported in protease immobilization studies [25]. At 850
C, the immobilized protease from AS#7 strain retained
5. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 90 | Page
~121% of its activity while free enzyme could retain 70% activity. The thermostability of enzyme increased
very significantly after entrapment. This probably reflects the fact that entrapped enzyme is not chemically
modified but remain in its native form in the gel matrix. Another reason, for this high optimum temperature of
immobilized protease from AS#7 strain may be high reaction rate of immobilized enzyme due to which reaction
is completed within a few minutes and the risk of denaturation was minimal [23]. Similar result was also
reported by Sharma et al, [25] with protease immobilized in Ca-alginate capsules.
Reusability of immobilized protease -
The activity of entrapped enzyme was assayed for the three cycles with casein as a substrate, in order to
find out the reusability of the entrapped enzyme. The entrapped enzyme showed 78% and 83% activity during
the second reuse and 78% and 80% activity on its third use, respectivety for protease from AS#6 and AS#7
strain. This decrease in activity was due to leakage of enzyme from the beads. In another study of entrapped
enzyme during forth cycle activity is completely lost. [20].
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Table -1Partial purification of crude protease from Aspergillus As # 6 strain by ammonium sulphate
precipitation.
Purification
steps
Total
enzyme
activity
(U)
Total
protein
(mg)
Specific
activity
U/mg
Fold
Purification
Yield
(%)
Crude enzyme 298 99.4 3.0 1 100
Ammonium
sulphate
fractionation
153 6.8 22.5 7.5 51
Table-2Partial purification of crude protease from Aspergillus As # 7 strain by ammonium sulphate
precipitation.
Purification steps Total
enzyme
activity
(U)
Total
protein
(mg)
Specific
activity
U/mg
Fold
Purification
Yield
(%)
Crude enzyme 385 98.2 3.9 1 100
Ammonium
sulphate
fractionation
165 2.2 75 19.2 42.9
7. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 92 | Page
8. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 93 | Page
9. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 94 | Page
Fig: 11 Effect of different temperature on protease activity from as#6
strain
10. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 95 | Page
Fig : 12 Effect of different temperature on protease activity from As#7
11. Kinetic study of free and immobilized protease from Aspergillus sp.
www.iosrjournals.org 96 | Page
Fig : 16 Effect of different temperature on immobilized protease activity from As#7 strain
Fig : 15 Effect of different temperature on immobilized protease activity from As#6 strain