This document discusses the immobilization of α-amylase enzyme on mesoporous silica (KIT-6) and palm wood chips (PWC) for starch hydrolysis. α-Amylase was immobilized on KIT-6 and PWC by physical adsorption. Activity tests showed that glucose concentration, and thus enzyme activity, increased with increasing enzyme concentration for both supports up to 0.5 v/v. KIT-6 showed superior performance compared to PWC, with a maximum glucose to support ratio of 48 for KIT-6 versus 0.6 for PWC. KIT-6 also demonstrated better thermal stability than PWC. The high performance of KIT-6 is attributed to
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.
Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
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
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).
Production and optimization of lipase from candida rugosa using groundnut oil...eSAT Publishing House
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.
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.
Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
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
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).
Production and optimization of lipase from candida rugosa using groundnut oil...eSAT Publishing House
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.
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.
Production of Amphiphilic Surfactant Molecule From Saccharomyces Cerevisiae M...inventionjournals
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.
Cellulase (Types, Sources, Mode of Action & Applications)Zohaib HUSSAIN
Cellulase is a class of enzyme that catalyzes the cellulolysis i.e., hydrolysis of cellulose. Celulase is a multiple enzyme system consisting of endo – 1, 4 –β–D – glucanases and exo – 1, 4 –β– D – glucanases along with cellobiase (β– D – glucosideglucano hydrolase).
Types of Cellulases
On the basis of fractionation studies on culture filtrate have demonstrated that, there are ‘three’ major types of enzymes involved in the hydrolysis of native cellulose to glucose, namely: Others are produced by the some animals and plants.
Amylase is an enzyme that catalyzes the hydrolysis of starch into sugars.
Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion. Foods that contain large amount of starch but less amount of sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase degrades some of their starch into sugar.
α- amylase is a protein enzyme that hydrolyses alpha bonds of large, alpha - linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is a major form of amylase found in humans and other mammals.
Many of the enzymes used in the industries are extracellular derived from microorganisms. Among various extracellular enzymes, alpha amylase ranks first in terms of commercial exploitation.
Bacteria and fungi secrets amylases to the outside of the cells to carryout extracellular digestions when they have broken down the soluble starch, the soluble end products such as Glucose or Maltose are absorbed into their cells.
The industrially important Bacillus strains which are extensively used to produce alpha amylase, are, B. licheniformis, B. subtilis etc. B. amyloliquefaciens
Bacillus licheniformis is a Gram-positive endospore forming organism that can be isolated from soils and plant material all over the world.
This organism is used extensively for large-scale industrial production of exoenzymes as it can secrete large quantities of proteins of up to 20–25 g/l.
The use of the submerged culture is advantageous because of the ease of sterilization and its process control.
The objective of this work was to study the pattern and the comparison of α-amylase production by using two strains of Bacillus licheniformis, MTCC 2617 and MTCC 2618 using four different substrates starch, rice, wheat and ragi powder as carbon source.
Ragi or finger millet is round, soft yet firm and rich brown in color. It is probably the only edible solid you are advised to swallow not chew. A gram of ragi has 72% carbohydrate, 3.6% fiber, 7.3% of protein, vitamin B and a good combination of minerals.
Pharmaceutical Biotechnology Research Presentation : Recombinant Streptokinase
Dr. Godfrey Mazhandu
Professor Peivand Pirouzi Inc. -
Copyright 2015 - Professor Peivand Pirouzi Inc., International Corporate Training, Canada
All rights reserved
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.
Production of Amphiphilic Surfactant Molecule From Saccharomyces Cerevisiae M...inventionjournals
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.
Cellulase (Types, Sources, Mode of Action & Applications)Zohaib HUSSAIN
Cellulase is a class of enzyme that catalyzes the cellulolysis i.e., hydrolysis of cellulose. Celulase is a multiple enzyme system consisting of endo – 1, 4 –β–D – glucanases and exo – 1, 4 –β– D – glucanases along with cellobiase (β– D – glucosideglucano hydrolase).
Types of Cellulases
On the basis of fractionation studies on culture filtrate have demonstrated that, there are ‘three’ major types of enzymes involved in the hydrolysis of native cellulose to glucose, namely: Others are produced by the some animals and plants.
Amylase is an enzyme that catalyzes the hydrolysis of starch into sugars.
Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion. Foods that contain large amount of starch but less amount of sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase degrades some of their starch into sugar.
α- amylase is a protein enzyme that hydrolyses alpha bonds of large, alpha - linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is a major form of amylase found in humans and other mammals.
Many of the enzymes used in the industries are extracellular derived from microorganisms. Among various extracellular enzymes, alpha amylase ranks first in terms of commercial exploitation.
Bacteria and fungi secrets amylases to the outside of the cells to carryout extracellular digestions when they have broken down the soluble starch, the soluble end products such as Glucose or Maltose are absorbed into their cells.
The industrially important Bacillus strains which are extensively used to produce alpha amylase, are, B. licheniformis, B. subtilis etc. B. amyloliquefaciens
Bacillus licheniformis is a Gram-positive endospore forming organism that can be isolated from soils and plant material all over the world.
This organism is used extensively for large-scale industrial production of exoenzymes as it can secrete large quantities of proteins of up to 20–25 g/l.
The use of the submerged culture is advantageous because of the ease of sterilization and its process control.
The objective of this work was to study the pattern and the comparison of α-amylase production by using two strains of Bacillus licheniformis, MTCC 2617 and MTCC 2618 using four different substrates starch, rice, wheat and ragi powder as carbon source.
Ragi or finger millet is round, soft yet firm and rich brown in color. It is probably the only edible solid you are advised to swallow not chew. A gram of ragi has 72% carbohydrate, 3.6% fiber, 7.3% of protein, vitamin B and a good combination of minerals.
Pharmaceutical Biotechnology Research Presentation : Recombinant Streptokinase
Dr. Godfrey Mazhandu
Professor Peivand Pirouzi Inc. -
Copyright 2015 - Professor Peivand Pirouzi Inc., International Corporate Training, Canada
All rights reserved
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Production of α-amylase using new strain of Bacillus polymyxa isolated from s...IOSR Journals
In this study, a new amylase producer strain was isolated from sweet potato tuber. This strain was able to grow at 37 °C and produce α-amylase in high quantity compared to other standard strain cultures. In the first part, cultivation in shake flask in standard medium was carried out to give complete information about the growth and production kinetics of this strain. The results clearly demonstrate that the isolated strain is able to production α-amylase in submerged culture with concentration up to 2050 kat/L after 20 h cultivation. Furthermore, medium optimization was carried out by changing the starch concentration and cell cultivation in medium of mixed carbon source (composed of starch and glucose of ratio 15:5 g/g) to enhance the production process and to increase the growth rate. The volumetric and specific α-amylase production in this optimized medium were 4550 kat/L and 1060 kat/g, respectively. Further improvement in enzyme production process was achieved by scaling up the process from shake flask to 3-L stirred tank bioreactor under non-oxygen limiting condition. The maximal volumetric and specific α-amylase productions in bioreactor batch culture were 5210 kat/L and 1095kat/g, respectively, after only 14 h cultivation
Isolation of Yeasts from Raisins and Palm-Juice and Ethanol Production in Mol...Shafkat Shamim Rahman
The alternative fuels are expected to satisfy the progressive demand for energy on the wake of the negative effects of fossil fuel on the atmosphere and resultant universal warming. In this study two ethanol fermenting Saccharomyces cerevisae were isolated from Palm juice and Raisins. Both isolates were grown in Yeast extract Peptone Dextrose (YEPD) medium and characterized for alcoholic fermentation using molasses medium and optimized for pH, thermo-, osmo-, ethanol tolerance and sugar concentration. Results showed for ethanol fermentation, 31°C temperature, 6.01 pH and 6.50% sugar concentration is the prime condition. Raisin-isolate emerged as highly thermophilic and stress tolerant in nature. Under optimized conditions, S. cerevisae isolated from Palmjuice produced 9.85% of ethanol in the medium. Creation of ethanol through fermentation appears to be a potential other fossil fuel and can be used as exclusive fuel in vehicles with dedicated engines or in fuel blends.
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
Production and Purification of Amylase from Bacillus subtilis Isolated from SoilDr. Amarjeet Singh
In spite of progress in biotechnology and
enzymology, the enzymes have been industrialized in recent
years for the mounting up the product development in
various arena. The ultimate goal of this study comprises the
production and purification the amylase enzyme from the
bacterial strain. A powerful amylase producer, Bacillus
subtilis ISOLATE-4 was isolated, screened and identified
from the soil sample. In order to produce extracellular
amylase, various physico-chemical parameters were
optimized. During optimization, the maximal production of
amylase by the isolate at 48 hrs of incubation in 100 rpm was
found to be 6.93U/ml, 5.94U/ml, 6.0U/ml at 45ºC, pH 6 with
1% substrate concentration respectively. Ammonium
sulphate fractionation was done for rapid precipitation of the
amylase at a concentration of 60% and exposed to dialysis
showed the 25% purification fold of an enzyme. The dialyzed
product was further subjected to DEAE-Cellulose column
chromatography resulted in an increase up to 75%
purification fold than crude enzyme. The amylase enzyme
might be suitable for the liquefaction of starch, detergent,
textile and several additional industrial applications.
A study with enzymatic membrane reactor for conversion of lactose in to galac...Pallavi Kumari
The formation of galacto-oligosaccharides (GOS) from lactose by commercially available Biolacta FN5 (β- galactosidase, EC 3.2.1.23) derived from Bacillus circulans was studied under immobilized enzyme condition. The present work utilizes hydrophobic membrane (0.22 m pore size) for immobilization of enzyme. Experiments were conducted in a three compartment cell. The middle compartment (~25 mL) being separated by immobilized membranes was utilized for feed lactose solution; whereas, adjacent compartments were filled with distilled water. The reacted mixture solution was analyzed for tri-, tetra- and penta- forms of GOS which depended on varying amounts of initial lactose (ILC) and enzyme concentrations. Total GOS formation increased from 7 to 28% for ILC from 50 to 200 g/L. However, tri-saccharide was the major (67%) in comparison to tetra (27%) and penta (6%) forms of GOS. There was marginal difference of GOS formations while comparing the result (GOS yield) under both free (~30%) and immobilized (~28%) conditions.
Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
Comparative Ethanol Productivities of Two Different Recombinant Fermenting St...IJERA Editor
Production of biofuel such as ethanol from lignocellulosic biomass is a beneficial way to meet sustainability and energy security in the future. The main challenge in bioethanol conversion is the high cost of processing, in which enzymatic hydrolysis and fermentation are the major steps. Among the strategies to lower processing costs are utilizing both glucose and xylose sugars present in biomass for conversion. An approach featuring enzymatic hydrolysis and fermentation steps, identified as separate hydrolysis and fermentation (SHF) was used in this work. Proposed solution is to use “pre-processing” technologies, including the thermal screw press (TSP) and cellulose-organic-solvent based lignocellulose fractionation (COSLIF) pretreatments. Such treatments were conducted on a widely available feedstock such as source separated organic waste (SSO) to liberate all sugars to be used in the fermentation process. Enzymatic hydrolysis was featured with addition of commercial available enzyme, Accellerase 1500, to mediate enzymatic hydrolysis process. On average, the sugar yield from the TSP and COSLIF pretreatments followed by enzymatic hydrolysis was remarkable at 90%. In this work, evaluation of the SSO hydrolysate obtained from COSLIF and enzymatic hydrolysis pretreaments on ethanol yields was compared by fermentation results with two different recombinant strains: Zymomonas mobilis 8b and Saccharomyces cerevisiae DA2416. At 48 hours of fermentation, ethanol yield was equivalent to 0.48g of ethanol produced per gram of SSO biomass by Z.mobilis 8b and 0.50g of ethanol produced per gram of SSO biomass by S. cerevisiae DA2416. This study provides important insights for investigation of the source-separated organic (SSO) waste on ethanol production by different strains and becomes a useful tool to facilitate future process optimization for pilot scale facilities.
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.
Effect of Various Parameters on the Growth and Ethanol Production by Yeasts I...Shafkat Shamim Rahman
Two ethanol fermenting Saccharomyces cerevisiae were isolated from date juice and grapes and grown in YEPD medium. They were characterized for alcoholic fermentation using sugarcane molasses and their growth conditions were optimized with respect to pH and sugar concentration. Results revealed a temperature of 30ºC, pH 6.0 and 6.5% sugar concentration as optimum for fermentation. Stress tolerance tests showed that date juice isolate was highly tolerant to temperature, pH and high ethanol concentration in the medium. Under optimized conditions, S. cerevisiae isolated from date-juice produced 7.75% of ethanol in molasses as estimated by Conway method.
Isolation and Characterization of Thermostable Protease Producing Bacteria fr...IOSR Journals
This study is a search for potential thermostable protease producing strain. Among nine protease
producing strains screened from soap industry effluent, one was selected as promising thermostable protease
producer and identified as Bacillus subtilis. The activity of the protease produced by this organism is stable up
to 70ºC. The optimum yield was achieved after 48 hours of culture, at 65ºC with the pH 8.0. The maximum
protease activity was observed at 65ºC and at pH 8.0.
Cassava Retting Water An Alternative Source for Industrial Cellulase EnzymeYogeshIJTSRD
Cassava fermentation is one of the teaming businesses in almost all the tribes of Nigeria. Among all the methods of cassava processing to food, fermentation is the most used. This produces foul smelling waste water that causes environmental pollution to man and animals. The retted cassava water was checked for cellulase enzyme activity to see if it can be a source of cellulase for used in food, paper, textile and other industries. The aim of this work is to seek a way of utilizing the waste water as source of enzymes as this will reduce the importation of these enzymes and make them available always. Cassava tubers were peeled, cut into cylindrical portions of about 3 5 cm and washed. Two hundred grams of the washed tubers were soaked in 5 liters of water and allowed to ret. The retting water was analyzed daily for titratable acidity, cyanide content, pH, cellulase activity and the microbial flora were isolated and identified. Results showed that titratable acidity rose from 0.20 to 2.76 mg g and cyanide content increased from 0.28 to 4.69 mg ml while pH fall from 7.2 – 6.0 tending acidic. Retting started on the 2nd day and complete retting was achieved on the 4th day. ß glucosidase activity rose from 0.05 to 8.0 µ mol, Filter paper activity increased from 0.06 to 7.5 µ mol and carboxyl methyl cellulase CMC activity increased from 0.05 to 7.7 µ mol. Ten organisms Aspergillus fumigatus, Rhizopus stolonifer, Bacillus subtilis, Candida utilis, Citrobacter sp, Enterobacter aerogenes, Lactobacillus coryneformis, Saccharomyces cerevisiae, Staphylococcus aureus, Streptococcus feacalis were isolated from the retting water. Daily increase in the enzyme activities showed that cassava retting when done in a large scale can yield large quantity of enzymes. This will reduce the importation of industrial enzymes and reduce the environmental pollution caused by the waste water. Umeh, S. O. | Nwiyi, I. U. | Dimejesi, S. A. | Ikele, M. O. | Ugwu, C. H. "Cassava Retting Water: An Alternative Source for Industrial Cellulase (Enzyme)" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd43889.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/43889/cassava-retting-water-an-alternative-source-for-industrial-cellulase-enzyme/umeh-s-o
Similar to Immobilization of α amylase on mesoporous silica kit-6 and palm wood chips for starch hydrolysis (20)
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Immobilization of α amylase on mesoporous silica kit-6 and palm wood chips for starch hydrolysis
1. Chemical and Process Engineering Research www.iiste.org
ISSN 2224-7467 (Paper) ISSN 2225-0913 (Online)
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Immobilization of α- Amylase on Mesoporous Silica KIT-6 and Palm
Wood Chips for Starch Hydrolysis
M. A. USMAN*, V. I. EKWUEME, T. O. ALAJE, M. T. AFOLABI AND S. O. BOLAKALE
Department of Chemical Engineering, University of Lagos, Lagos 101017, Nigeria
*Corresponding author: mawwal04@yahoo.com; 2348023186445
Abstract
α-Amylase was immobilized on highly ordered mesoporous silica KIT-6 and palm wood chips (PWC) by physical
adsorption. The activity of the immobilized enzyme for starch hydrolysis was investigated at different solution pH
and enzyme concentration for both supports. The thermal stability of the immobilized enzyme was also investigated
for both supports. The result indicates that the maximum activity, in terms of glucose concentration, for both supports
was observed close to the isoelectric point (PI = 7.00) of the enzyme. Activity increased with increasing
concentration of the enzyme solution for both supports. The glucose to support ratio of 48 was obtained for KIT-6
compared to a value of 0.6 obtained for PWC. KIT-6 shows superior thermal stability. The exceptional performance
of KIT-6 relative to PWC can be ascribed to its high adsorptive capacity as well as its excellent mass transfer
characteristics arising from its very high surface area, large pore diameter and highly ordered pores.
Keywords: Mesoporous silica, KIT-6, Palm wood chips, Enzyme immobilization, α-amylase, Starch hydrolysis
1. Introduction
Enzymes are biocatalysts that are gradually making in-roads in various fields of chemical engineering, where
chemical synthesis routes are being replaced by enzymatic ones. Enzymatic routes offer advantages in terms of
selectivity with its concomitant high yield and exclusivity towards the desired product (Lilly, 1994). Commercial
application of enzymes is however fraught with the difficulty of separation from the solution and their inactivation
by organic solvent and extreme pH or temperature. Immobilized enzymes are being increasingly used owing to their
ease of separation and enhanced thermal or pH stability (Wang et al., 1992; Leckband and Langer, 1991; Pierre and
Crichton, 1988). The immobilized enzyme molecules may also be stabilized against denaturing agents that promote
unfolding processes that can destroy the active site (Mozhaev, 1993). Thus, immobilization reduces loss of enzyme
and offers the opportunity to use a continuous reactor with a reuse of the enzyme for many reaction cycles to give an
economic advantage (Posorske, 1984).
Amylases are endo-enzymes which hydrolyze starch molecules to give diverse products including dextrins and
progressively smaller polymers composed of glucose units. These enzymes are of great significance in present day
biotechnology with applications ranging from food, baking, brewing, fermentation, detergents, textile designing, and
paper industries to analysis in medicinal and clinical chemistry (Ahmed et al., 2008). Like any other enzyme, α-
amylase has to be immobilized to improve its industrial applications.
Various solid support materials have been studied for the immobilization of enzymes such as clay/modified clays
(Naidja and Huang, 1997,1996; Lopez Santin et al., 1983), silica (Pierre and Crichton, 1988; Martins and Cruz,
1987), zeolite (Pifferi et al., 1982), amorphous aluminium phosphate (Felipa et al., 1998), zirconia (Reshmi et al.,
2007), calcium alginate beads (Roy and Gupta, 2004), palm wood chips (Bello and Ogunbayo, 1986, 1993, 2011),
mesoporous silicas MCM-41 and SBA-15 (Pandya et al., 2005; Zou et al., 2010).
Palm wood chips (PWC) are medium-sized solid material made by cutting or chipping large pieces of wood. Its main
constituent includes cellulose and lignin. PWC offer advantages in terms of high adsorptive capabilities and retention
of enzymes for a long time (Bello and Ogunbayo, 1986, 1993; Egwin and Oloyede, 2008). Enzymes have been
successfully immobilized on PWC and used for the hydrolysis of starch (Bello and Ogunbayo, 1993; Egwin and
Oloyede, 2008).
Mesoporous silicas are interesting solids for studying enzyme immobilization due to their relatively uniform pore
structure, large surface area and biocompatibility. It is well known that pore size, pore volume and pore structure of
mesoporous materials have influence on the immobilization of enzymes (Wei et al., 2008). However, 1-D and 2-D
structure, lower pore size and pore volume of the early mesoporous materials such as MCM-41 and SBA-15 restrict
enzyme diffusion and transportation, thus limiting their use in the field of enzyme immobilization. Mesoporous silica
KIT-6 has unique 3-D interconnected network which provides a highly open porous host with an easy and direct
access for guest species, thus facilitating inclusion or diffusion throughout the pore system without pore blockage
(Guo et al., 2010).
2. Chemical and Process Engineering Research www.iiste.org
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The main aim of this work was to investigate the activity and stability of α-amylase immobilized on ordered
mesoporous silica KIT-6 for starch hydrolysis in comparison with the performance of α-amylase immobilized on
palm wood chips (PWC). Performance was expressed in terms of concentration of glucose produced.
2. Materials and Methods
2.1 Materials
α-amylase (Brand: Termanyl, 120 L, Type L) in sodium phosphate buffer, palm wood chips, dinitrosalicyclic acid,
corn starch powder, acetic acid, sodium hydroxide, distilled water, acetic acid, aluminium foil, filter papers.
Mesoporous silica KIT-6 was synthesized and characterized as reported in our previous work (Usman et al., 2012).
Other reagents used in this work were of analytical grade and used as-received.
2.2 Preparation of Enzyme
Two sets of enzyme samples were prepared for this study. The first set of five samples were prepared by adding 10,
20, 30, 50, and 60 ml of the enzyme in sodium phosphate buffer to 90, 80, 70, 50 and 40 ml of distilled water
respectively, in a conical flask, to give concentration range of 0.1 – 0.6 v/v. The pH of each solution was 7.00 ± 0.4.
The second set of three samples was prepared at varied pH but constant concentration of 0.5 v/v. This was achieved
by adding 2 ml of 0.1 M, 0.5 M NaOH to the 1st and 2nd conical flask containing enzyme of concentration 0.5 v/v
with corresponding pH 8.20 ± 0.2, 10.00 ± 0.40 respectively, 2 ml of 0.5 M acetic acid with pH 6.7 ± 0.1 in the 3rd
conical flask.
2.3 Evaluation of Enzyme Activity
The prepared enzyme solutions were immobilized on PWC and KIT-6 and used for the hydrolysis of starch. For the
immobilization on PWC, 2 g of wood chips each was used to immobilize the enzyme at concentrations of 0.1 – 0.6
v/v and at solution pH of 7.04, 8.40, 10.40 and 6.80 (all at a constant concentration of 0.5 v/v), which gave a total
setup of 10 conical flasks. The resulting mixture was covered with aluminum foil, continuously shaken in a water
bath at 100 shakes/min at 27.2 o
C for 4 hrs. The treated PWC were washed three times with distilled water to remove
excess enzyme on the surface and subsequently used for starch hydrolysis.
For the immobilization on KIT-6, 50 mg of the highly ordered mesporous siliceous material each was used to
immobilize the enzyme at concentrations 0.1v/v, 0.3v/v, 0.5 v/v, 0.60 v/v (all at PH 7.00) and solution PH of 6.70,
7.02, 8.20 and 10.20 at constant enzyme concentration of 0.5 v/v, these gave a total set of 8 conical flasks. The
resulting mixture was covered and also agitated. After 4Hrs, each KIT-6 setup was centrifuged at 3500 rpm for 10
mins, cooled in ice water at intervals of 2 mins. The liquid on top was decanted and the resulting loaded material was
dried at room temperature and was subsequently used for starch hydrolysis.
The starch hydrolysis was carried out by preparing a starch solution of 0.025 g/ml of distilled water. 2 g (2000 mg of
the loaded wood chips) was immersed in 50 ml of starch solution (40:1) and 40 mg of the loaded KIT-6 organosilica
in 1 ml of starch solution (40:1) and hydrolysis in each flask allowed for 40 mins at 27.20
C and 100 shakes/min in a
water bath.
2.4 Thermal Stability Experiment
The prepared enzyme sample, 0.1 v/v concentration and pH 7.00, was immobilized on PWC and KIT-6 and used for
this study. For the KIT-6, 350mg of the carrier was immersed in a conical flask containing 100 ml of the enzyme
solution for 4 hours. After immobilization, the solution was centrifuged at 3500 rpm for 5 minutes to separate the
immobilized KIT-6 from the solution. The KIT-6 was left to dry in air overnight. For the PWC, the carrier was boiled
in water at 80o
C for 30 minutes. The boiled chips were then immersed in 100 ml of the enzyme solution for 4 hours.
The chips were then removed and rinsed with water to remove excess enzyme.
Hydrolysis of starch using immobilized α-amylase on PWC was performed according to the following procedure.
2 %w/v of starch solution was prepared by dissolving 4 g of starch in 200 ml of hot water. 70 ml of the prepared
starch solution was placed in a conical flask and 2 g of the immobilized α-amylase on PWC added to it. The flask
was immersed in the water bath at 30o
C for 40 minutes and agitated at the speed of 100 shakes per minute for
hydrolysis to take place. Thereafter, the PWC were removed to stop the hydrolysis. The procedure was repeated at
temperatures of 40, 50 and 60o
C respectively.
Hydrolysis of starch using immobilized α-amylase on KIT-6 was carried out as follows. 2 % w/v of starch solution
was prepared by dissolving 4 g of starch in 200 ml of hot water. 5 ml of the prepared starch solution was placed in a
conical flask and 20 mg of the immobilized KIT-6 was added. The mixture was heated to 30o
C and agitated using a
magnetic stirrer for 40 minutes. Hydrolysis was stopped by heating the solution in boiling water. The procedure was
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repeated at temperatures of 40 and 50 o
C.
2.5 Analysis
The dinitrosalicyclic acid (DNSA) method was used for estimating the reducing sugar content (glucose) of the
product of the experimental runs. DNSA was mixed with the hydrolysed product( P) in the ratio of DNSA:P of 1:2
and after heating in the water bath for 5 mins , the yellow colour of the mixture change to deep red; an indication of
the reaction between DNSA and the hydrolysed product. The maximum absorbance of each sample was measured in
a colorimeter at 660 nm and the corresponding pure glucose concentration was obtained from a standard glucose
curve at 660 nm.
3. Results and Discussion
3.1 Evaluation of Enzyme Activity
3.1.1 Effect of Enzyme Concentrations.
As shown in Figure 1, there is an initial rise in glucose concentration with increasing enzyme concentration for both
KIT-6 and PWC, though sharper for KIT-6. This indicates a high affinity of the supports for α-amylase molecules.
With increasing α-amylase concentration, the glucose concentration increases which means that the amount of α-
amylase absorbed on both supports increase.
However, the glucose concentration attains a maximum value of 1.91g/l for KIT-6 and 1.24g/l for PWC for both
supports at 0.50 v/v. This trend may be due to the fact that the protein molecules may be adsorbed on the solid
supports in various distinct orientations (Vinu et al., 2005). At low enzyme concentrations, the α-amylase molecules
may be adsorbed with a side-on- type configuration perpendicular to the supports. When the enzyme concentration is
high, the molecule may be adsorbed with an end-on-type configuration that helps the molecule land close to each
other, which reduce the increasing electrostatic repulsion between the protein molecules resulting in higher α-
amylase adsorption. Moreover, the high concentration of the enzyme helps close packing because of the decreased
hydrophobic interactions between the protein and the support upon adsorption of some α- amylase molecules.
Therefore, the maximum glucose concentration was observed at 0.5v/v for both supports.
Figure 1: Glucose Concentration ( g/l) vs Enzyme Concentration (v/v) .
3.1.2 Effect of Support Material
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To ensure that the glucose concentration will be a function of only the acivity of the enzyme, the temperature was
kept constant at 27.20
C. As shown in figure 2, as the concentration of the enzyme increases, the glucose/support ratio
of both support materials increases before attaining a near contant value at 0.5v/v. Interestingly, the trend shows that
at an enzyme concentration of 0.1 v/v, the ratio is 36:1 this implies that 1g of KIT-6 (with immobilized enzymes)
will be required to produce 36g of glucose, while for PWC (with immobilized enzymes) 1g will be producing just
0.51g of glucose. Generally 40mg of α-amylase/KIT-6 produced 1.44g glucose/l at an enzyme concentration of
0.1v/v, while 2g α-amylase/PWC produced 1.02g glucose/l. At an enzyme concentration 0.3 v/v, more α-amylase
molecules are active. 40mg of α-amylase/KIT-6 produced 1.76g of glucose/l (increment of 0.32g) and 2g of wood
chips produced 1.16g of glucose (increment of 0.14g). Finally, at 0.5 v/v, 40mg KIT-6 produced 1.91g of glucose (an
increment over the previous value which is twice that of PWC) and 2g of PWC produced 1.24g of glucose. Hence,
activity increases as enzyme concentrations increases up to 0.5 v/v and KIT-6 is shown to be a superior support
compared with PWC.
The reason for this high adsorption capacity and activity of the enzyme on KIT-6 is due to its well- ordered, large
pore diameter and pore volume. Besides, it was explained earlier that KIT-6 has a 3-D pore network with two
intersecting pore systems which allows the α-amylase to access the adsorption sites from all the three dimensions,
unlike PWC which is a relatively poorly ordered support.
It was also observed during experiment that the absorbances in the 1st and 3rd hours of starch hydrolysis are
approximately the same for KIT-6, while an increase was observed when PWC were used. Thus, hydrolysis of starch
using α-amylase on KIT-6 support is accomplished with a shorter time than with PWC. This advantage has
commercial appeal and lends credence to the earlier assertion that KIT-6 support are more elegant house and better
than PWC.
Figure 2: Glucose/support ratio vs enzyme concentrations for KIT-6 and PWC.
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Figure 3: Effect of solution pH for both supports (PWC and KIT-6).
3.1.3 Influence of Solution pH
Figures 3 show that the glucose concentration increases with increasing solution pH; from pH 6.80 reaches a
maximum at 7.04 for wood chips and pH 6.70 reaches a maximum at 7.02 for KIT-6 and then decreases in both
cases.
Their corresponding glucose concentrations are1.23g/l and 1.88g/l for wood chips and KIT-6 respectively. These
amounts are recorded near the isoelectric point of α-amylase in Na2PO4 buffer pH=7.00. This trend also shows that
α-amylase molecules work relatively well in a acidic medium (Acetic acid) than in basic meduim. At the isoelectric
point, the net charge of the enzyme is low, coulombic repulsive force between and (or) within the protein molecule
will be minimal and closer packing of the protein molecule will be minimal (Vinu et al, 2008).
Far above or far below this point, the glucose concentration significantly decreases and this negatively affects the
stability and re-usability of the enzyme for starch hydrolysis i.e. the activity of the enzyme would significantly
decrease in a highly acidic and highly basic medium.
3.2 Thermal Stability
From the Figures 4, it is seen that there is a high increase in glucose concentration for enzyme immobilized on both
PWC and KIT-6 as the temperature increases from 300
C-400
C. Likewise, there is also an increase as the temperature
increases from 400
C-500
C. The increase in concentration shows a drop of 65.3% -13% for PWC as opposed to
71.6%-65.2% for KIT-6. The reason for the higher drop obtained for wood chips is as a result of its pore structure,
which favours the leaching of the enzyme, thus reducing its activity over time, unlike KIT-6 which maintains the
attachment of the enzyme over time. Thus, it can be said that KIT-6 is a better support than PWC.
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Figure 4: Effect of temperature on enzyme activity
4. Conclusion
The activity of α-amylase during starch hydrolysis has been studied with KIT-6 and PWC as supports at different
enzyme concentrations and solution pH. It is evident that KIT-6 is a better support compared to PWC at these
operating conditions. The maximum glucose concentration was 1.91g/l for KIT-6 and 1.24g/l for wood chips at 0.5
v/v when varying enzyme concentrations at constant solution pH = 7.00 and 27.20
C. At varying solution pH, the
maximum activity was observed very close to the isoelectric point of α-amylase (pH = 7.00), 7.04 for PWC and 7.02
for KIT-6 with their respective glucose concentrations 1.23g/l and 1.88g/l. More enzymes were immobilized on KIT-
6 than wood chips due to its very high surface area and well- ordered, large pore, interpenetrating and interconnected
channels. This gives room for a higher adsorption as well as better mass transfer characteristics thus producing a
higher glucose/support ratio. Thermal and mechanical stability study also confirms the superiority of KIT-6 over
PWC.
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submission. Prospective authors of IISTE journals can find the submission
instruction on the following page: http://www.iiste.org/Journals/
The IISTE editorial team promises to the review and publish all the qualified
submissions in a fast manner. All the journals articles are available online to the
readers all over the world without financial, legal, or technical barriers other than
those inseparable from gaining access to the internet itself. Printed version of the
journals is also available upon request of readers and authors.
IISTE Knowledge Sharing Partners
EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open
Archives Harvester, Bielefeld Academic Search Engine, Elektronische
Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial
Library , NewJour, Google Scholar