A high alkaline protease producing bacterial strain was isolated and identified a local soil sample. The organism was gram positive and forms spore during adverse condition in the growth medium. After various tests it was suggested and the features agreed with the description of Bacillus subtilis. It was also identified as B. subtilis with 99.9% identity by API 50 CHB. The enzyme hydrolyses a number of proteins including azocasein which suggests that it is an extracellular alkaline protease. The experimentally determined isoelectric point was 5.1 and the optimal enzyme activity was at 60°C and at pH 8.5. The esterase preferentially hydrolyzed short-chain fatty acids. Native enzyme preparations typically showed a Michaelis constant (Km) and Vmax of 0.40mM and 12,200 U mg)-1, respectively. This microbial enzyme was partially purified by ammonium sulfate fractionation, dialysis, DEAE cellulose chromatography and electrophoretic analysis. Enzyme purity was tested by SDS-PAGE. Quantitative estimation has shown that 40mL of culture supernatant could dehair 2×1 cm of leather completely in 9 hours. In future the tanneries will use a combination of chemical and enzymatic processes. In practical applications, protease is a useful enzyme for promoting the hydrolysis of proteins and showing significant industrial applications.
Isolation and screening of haloalkaline protease producing bacteria from tann...eSAT Journals
Abstract
Twenty bacterial strains were isolated on selective milk agar plates (pH 9.0) from tannery solid waste on the basis of different
colony morphology. These strains exhibited variable haloalkaline protease activity and were tolerant to different concentration of
both chromate (350-1450 μg/ml) and NaCl (2-9%). Those having clearance zone greater than 20.0 mm were considered as
significant isolate. Out of twenty, nine strains were interestingly tolerant to high concentration of Cr(VI) (850-1450 mg/l) and
NaCl (6.5-9%) and exhibited vibrant clear zone diameter between 21-35 mm. All these isolates in this study were capable of
reducing Cr(VI) aerobically and the reduction values ranged between 50.0-76.0% after 16-20 h of growth. Further, significant
haloalkaline protease production was observed in these bacteria within 24 h under wide temperature (25-45°C) and pH (8.0-10.5)
range. The potential of these strains to produce protease at higher rate in the presence of wheat bran as a cheap carbon source
and yeast extract or beef extract as a nitrogen source makes them a potential candidate for industrial applications and removal of
Cr(VI) and proteinaceous waste simultaneously from industrial waste of alkaline nature.
Key words: Tannery waste, chromate resistant, bacteria, haloalkaline, protease
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
PRODUCTION OF PROTEASE BY ALKALOPHILIC BACILLUSSUBTILIS IN BIOREACTOR AND ITS...AM Publications
The current studies were aimed at to investigate role of pH, dissolved oxygen for production protease in bioreactor by alkalophilic bacterium and application of saw dust for its purification. The production of proteolytic enzyme by Bacillus subtilis IC-5 started as pH of medium falls to 9 and reached to maximum at pH 7 i.e., 4400 Uml-1 . Likewise dissolved oxygen decreased in the medium as the protease production progresses. Saw dust was successively utilized for partial purification of protease. The partial purification of protease increased the specific activity to5.3 fold. The optimum pH and temperature for purified activity was 11 and 700C, respectively. The purified enzyme was stable up to pH 12 and 80oC.
Screening and Production of Protease Enzyme from Marine Microorganism and Its...iosrjce
Marine sediment samples were collected from the Gulf of Mannar, Mandapam coast to screen for
protease producing microbes. Among the five isolates screened only two isolates showed maximum proteolytic
activity with the zone of 21mm and 19mm respectively. Biochemical characterization of the isolates were
performed and identified as strain P2 belonged to Bacillus subtilis and strain P5 belonged to Bacillus
licheniformis. Both the strains have the ability to tolerate 7%Nacl concentration. The amount of protease
produced was expressed in microgram of tyrosine released under standard assay conditions. The total protein
content of crude enzyme extracts of Bacillus subtilis and Bacillus licheniformis were quantified which revealed
21.2mg/ml for strain P2 and 22.4mg/ml of protein content was presented by strain P5. The proteolytic bacteria
gave an optimum performance were both strains exhibited the enzymes stable at PH
7. In the present study
Bacillus subtilis showed a remarkable activity at 40ºC where as Bacillus licheniformis exhibited maximum
activity at 50ºC. Studies pertaining to carbon sources starch and lactose were utilized by Bacillus subtilis and
Bacillus licheniformis and maximum production was achieved. Among the different nitrogen sources tested
yeast extract induced maximum proteolytic activity where as ammonium sulphate was found to be the best
nitrogen sources for protease production. The crude enzyme was efficient to remove hair dye and blood stain by
Bacillus subtilis and Bacillus licheniformis
This articles is based on information regarding how to produce microbial enzymes, methods of enzyme purification including sources and application of microbial enzymes.
Isolation and screening of haloalkaline protease producing bacteria from tann...eSAT Journals
Abstract
Twenty bacterial strains were isolated on selective milk agar plates (pH 9.0) from tannery solid waste on the basis of different
colony morphology. These strains exhibited variable haloalkaline protease activity and were tolerant to different concentration of
both chromate (350-1450 μg/ml) and NaCl (2-9%). Those having clearance zone greater than 20.0 mm were considered as
significant isolate. Out of twenty, nine strains were interestingly tolerant to high concentration of Cr(VI) (850-1450 mg/l) and
NaCl (6.5-9%) and exhibited vibrant clear zone diameter between 21-35 mm. All these isolates in this study were capable of
reducing Cr(VI) aerobically and the reduction values ranged between 50.0-76.0% after 16-20 h of growth. Further, significant
haloalkaline protease production was observed in these bacteria within 24 h under wide temperature (25-45°C) and pH (8.0-10.5)
range. The potential of these strains to produce protease at higher rate in the presence of wheat bran as a cheap carbon source
and yeast extract or beef extract as a nitrogen source makes them a potential candidate for industrial applications and removal of
Cr(VI) and proteinaceous waste simultaneously from industrial waste of alkaline nature.
Key words: Tannery waste, chromate resistant, bacteria, haloalkaline, protease
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
PRODUCTION OF PROTEASE BY ALKALOPHILIC BACILLUSSUBTILIS IN BIOREACTOR AND ITS...AM Publications
The current studies were aimed at to investigate role of pH, dissolved oxygen for production protease in bioreactor by alkalophilic bacterium and application of saw dust for its purification. The production of proteolytic enzyme by Bacillus subtilis IC-5 started as pH of medium falls to 9 and reached to maximum at pH 7 i.e., 4400 Uml-1 . Likewise dissolved oxygen decreased in the medium as the protease production progresses. Saw dust was successively utilized for partial purification of protease. The partial purification of protease increased the specific activity to5.3 fold. The optimum pH and temperature for purified activity was 11 and 700C, respectively. The purified enzyme was stable up to pH 12 and 80oC.
Screening and Production of Protease Enzyme from Marine Microorganism and Its...iosrjce
Marine sediment samples were collected from the Gulf of Mannar, Mandapam coast to screen for
protease producing microbes. Among the five isolates screened only two isolates showed maximum proteolytic
activity with the zone of 21mm and 19mm respectively. Biochemical characterization of the isolates were
performed and identified as strain P2 belonged to Bacillus subtilis and strain P5 belonged to Bacillus
licheniformis. Both the strains have the ability to tolerate 7%Nacl concentration. The amount of protease
produced was expressed in microgram of tyrosine released under standard assay conditions. The total protein
content of crude enzyme extracts of Bacillus subtilis and Bacillus licheniformis were quantified which revealed
21.2mg/ml for strain P2 and 22.4mg/ml of protein content was presented by strain P5. The proteolytic bacteria
gave an optimum performance were both strains exhibited the enzymes stable at PH
7. In the present study
Bacillus subtilis showed a remarkable activity at 40ºC where as Bacillus licheniformis exhibited maximum
activity at 50ºC. Studies pertaining to carbon sources starch and lactose were utilized by Bacillus subtilis and
Bacillus licheniformis and maximum production was achieved. Among the different nitrogen sources tested
yeast extract induced maximum proteolytic activity where as ammonium sulphate was found to be the best
nitrogen sources for protease production. The crude enzyme was efficient to remove hair dye and blood stain by
Bacillus subtilis and Bacillus licheniformis
This articles is based on information regarding how to produce microbial enzymes, methods of enzyme purification including sources and application of microbial enzymes.
Microbes, or microscopic organisms, are widely used in large-scale industrial processes. Microbes can be used to create biofertilizers or to reduce metal pollutants. Microbes can also be used to produce certain non-microbial products, such as the diabetes medication insulin, vaccines, etc. These slides will give insights into uses of microbes in production of enzymes, antibiotics, beverages, vitamins, vaccines, probiotics, etc
This PPT will provide the basic idea of Fermentation technology and it's use. The reference book is 'Pharmaceutical Biotechnology' by Giriraj Kulkarni.
Scope of Industrial Microbiology and BiotechnologyDr. Pavan Kundur
Industrial microbiology defined as the study of the large-scale and profit motivated production of microorganisms or their products for direct use, or as inputs in the manufacture of other goods.
Upon the evolution brought about in the fermentation technology resulted out into various methodologies for optimization of the product yield by economical consumption of the substrates. Eventually, these ventures led for the development of technologies classified into as Submerged and Solid State technologies and the latter one being the concept of interest whose detailed view will be provided in the following presentation
Production of cellulase and it's applicationRezwana Nishat
Cellulase is an enzyme that are found in livestock animals and herbivores' digestive system. It is also found in microbes system which is a great deal for researchers to study this enzymatic system furthermore. In this presentation, the production and the applications of this enzyme for biostoning of denim and cellulose nanofiber production have been studied.
UNIT-1 Introduction to biotechnology and enzyme immobilisation Brief introduc...Shyam Bass
(6th Sem B.Pharma Pharmaceutical Biotechnology)
UNIT-1 Introduction to biotechnology and enzyme immobilization Brief introduction to biotechnology, Enzyme biotechnology- methods of enzyme immobilization and applications, biosensors- working and applications of biosensors in pharmaceutical industries
The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!!
Mayur D. Chauhan
Microbes, or microscopic organisms, are widely used in large-scale industrial processes. Microbes can be used to create biofertilizers or to reduce metal pollutants. Microbes can also be used to produce certain non-microbial products, such as the diabetes medication insulin, vaccines, etc. These slides will give insights into uses of microbes in production of enzymes, antibiotics, beverages, vitamins, vaccines, probiotics, etc
This PPT will provide the basic idea of Fermentation technology and it's use. The reference book is 'Pharmaceutical Biotechnology' by Giriraj Kulkarni.
Scope of Industrial Microbiology and BiotechnologyDr. Pavan Kundur
Industrial microbiology defined as the study of the large-scale and profit motivated production of microorganisms or their products for direct use, or as inputs in the manufacture of other goods.
Upon the evolution brought about in the fermentation technology resulted out into various methodologies for optimization of the product yield by economical consumption of the substrates. Eventually, these ventures led for the development of technologies classified into as Submerged and Solid State technologies and the latter one being the concept of interest whose detailed view will be provided in the following presentation
Production of cellulase and it's applicationRezwana Nishat
Cellulase is an enzyme that are found in livestock animals and herbivores' digestive system. It is also found in microbes system which is a great deal for researchers to study this enzymatic system furthermore. In this presentation, the production and the applications of this enzyme for biostoning of denim and cellulose nanofiber production have been studied.
UNIT-1 Introduction to biotechnology and enzyme immobilisation Brief introduc...Shyam Bass
(6th Sem B.Pharma Pharmaceutical Biotechnology)
UNIT-1 Introduction to biotechnology and enzyme immobilization Brief introduction to biotechnology, Enzyme biotechnology- methods of enzyme immobilization and applications, biosensors- working and applications of biosensors in pharmaceutical industries
The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!!
Mayur D. Chauhan
Variation in trypsin inhibitor activity (TIA) and protein solubility within commercial soybean meals (SBM) is believed to affect animal performance. The objective of this research was to investigate the proteolytic effect of purified pancreatic trypsin/chymotrypsin and a purified protease from Nocardiopsis prasina on 9 commercial SBM samples varying in chemical composition, TIA and protein solubility characteristics. SBM was incubated with or without protease (pH 7, 3 hours, 40°C) and the supernatants were analyzed by SDS-page and for level of free soluble amino ends using the o-phthal-dialdehyde method. SDS-page analysis showed differences in the amount and composition of the soluble protein fraction of the SBM. The proteolytic efficiency of the lower dosages of pancreatic protease varied extensively between SBM batches whereas at the highest dose the efficiency was comparable for all SBM. The difference could not be explained by differences in protein solubility and TIA values as isolated variables, but the data strongly suggest that the hydrolysis of soya protein by pancreatic proteases depends on the commercial batch of SBM used. Incubation with N. prasina protease showed similar response for the different SBM, the main difference being a somewhat lower effect at high protease dose for one of the SBM batches, which might be in part explained by its distinct lower protein solubility
protease activity of extracellular enzyme produced by b. subtilis isolated fr...IJEAB
Background: Proteases produced by enzymatic method are more environments friendly than chemical process, and they have tremendous potential in the leather industry and in other several industries. In this study extracellular protease producing non pathogenic Bacillus subtilis was isolated from soil sample and relationship between sporulation and extracellular protease synthesis in large scale cultivation was studied. The enzyme was further characterized, purified, and tested for potential application. Result: The molecular weight of the protease was found to be ~30 KDa. Enzyme activity was checked on the presence of different metal ions and effectors. The enzyme was slightly modulated by MG++ ion, and significantly by Hg++ ion, while Zn++ ion slightly decrease the proteolytic activity. Sulfahydryl reagents, DTT slightly and β-ME significantly inhibit the enzyme. EDTA showed no effect on the enzyme suggesting that the enzyme might not be metalloprotease. PMSF, a known serine protease inhibitor was seen to totally inhibit the enzyme which indicates that the enzyme is a serine protease. The optimum enzyme activity was observed after 22 hours of incubation of B. subtilis at 37o C. Conclusions: Crude enzyme contains 285 units of enzyme which have direct dehairing activity. The enzyme was also seen to be able to remove blood and curry stain from clothes; making it a very promising candidate to be used in a leather and detergent industry. Apart from protease the bacterium was also seen to have lipase and collagenase activity. So, the bacteria are potentially good candidate for industrial application.
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.
Bacterial pigments have many applications in current day to day life. The pigments produced by chromobacteria can be used for various applications like dairy, pharmaceutical, and food etc. In this study, three types of pigments were isolated i.e. yellow from Xanthomonas sp., pinkish Red from Rhodotorula sp., and orange from Sarcina sp. Pigmented bacterial isolates were obtained from the soil samples and used for the pigment extraction study. We studied that the pigment producing bacteria and identified the color producing pigments. Soil samples from Pondicherry, Cuddalore, Chennai, and Andhra sea coast were collected and used for isolation of microbes producing pigments. Purification of extracted pigments were done by column chromatography, whereas identification and characterization of purified pigment done by UV-Visible spectrophotometry and GC/MS analysis etc. The pigment isolated from bacterial sp. were used for the antimicrobial activity, antioxidant, and anticancer & transformation studies. The bacterial extracts of carotenoid pigment extracted and used as natural colorants for food products and dying of cloth.
Key-words: - Soil samples, GC/MS analysis, UV-Visible spectrophotometry, Carotenoid, Pigment extraction
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.
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.
Production and optimization of lipase from candida rugosa using groundnut oil...eSAT Journals
Abstract The present work deals with the screening of microorganisms Candida rugosa NCIM 3467 and Penicillum citrinum NCIM 765 with different agro residues – rice bran, wheat bran, groundnut oil cake, coconut oil cake and sesame oil cake for maximum production of lipase. Among all the industrial residues, Groundnut oil cake supported the maximum lipase production by C.rugosa NCIM 3467. The physical factors such as fermentation time, temperature, pH, inoculum age, inoculum level, initial moisture content played a vital role in lipase production and further the yield was improved with the supplementation of carbon and organic nitrogen sources to the solid medium. At 5 days of fermentation, 32 °C, pH 6, 5 day old culture, 15% inoculum level and at 60% initial moisture content, lipase activity of 57.25 U/ml was obtained. Further the activity was raised to 63.35 U/ml by supplementing the substrate media with maltose (5%w/w) and peptone (3%w/w). Keywords: Candida rugosa, Pencillum citrinum, Solid state Fermentations, Lipase, Optimization and Characterization.
Phytochemical screening and antimicrobial studies of uapaca togoensis (pax) s...theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
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
Detection of Alpha-Amylase Activity from Soil Bacteriaiosrjce
Alpha-amylase is one of the industrial enzymes that hydrolyze starch molecules into polymers
composed of glucose units. The enzyme has potential application in a wide number of industrial processes such
as food, textile, paper, detergent, fermentation and pharmaceutical industries. Alpha-amylase can be produced
by microorganisms, plants or animals.
Aim: The aim of this study is to detect the activity of alpha-amylase from bacteria isolated from soil
environment.
Method: Soil samples were inoculated onto the media that are rich in nutrient that favour the growth of the
bacteria and incubated for 24 hours at 37oC after which the bacterial growth was detected in form of colonies.
In this study, bacterial species belonging to the genus Bacillus were identified through phylogenetic analysis
using 16s-ribosomal RNA sequencing for detection of the enzymatic activity. Effects of pH and temperature on
the enzymatic activity were observed using DNS activity assay method.
Results: Positive response to alpha-amylase activity by the soil bacteria was observed by the formation of clear
zone of inhibition shown by the colonies on the petri plates.
Conclusions: The optimal pH and temperature activities showed that the bacteria exhibit enzymatic activity at
mesophilic temperature and acidophilic or alkalophilic pH.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
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.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Microbial Production Of Alkaline Proteases And Evaluation Of Its Performances For Pretreatment Of Leather Industry
1. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
www.jmest.org
JMESTN42350702 3370
Microbial Production Of Alkaline Proteases And
Evaluation Of Its Performances For
Pretreatment Of Leather Industry
Uddin M. E.1
, Ahmad T.
1
, Sarkar M. K. I.2
, Azim D. A.
2
, Rahman S. S.3
, Islam M. S.
4
, Karim M. R.
5
, Rahman M.
M.
1
, Rahman M.
6
, Islam M. R.*1
1 Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh
2Department of Biochemistry, Primeasia University, Dhaka, Bangladesh
3
Department of Math and Natural Science (MNS), BRAC University, Dhaka, Bangladesh
4
Department of Applied Nutrition and Food Technology, Islamic University, Kushtia, Bangladesh
5
Department of Microbiology, Chittagong University, Chittagong, Bangladesh
6Department of Biochemistry and Molecular Biology, Dhaka University, Dhaka, Bangladesh
*
Corresponding author: Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia,
Bangladesh, E. mail: rezwaniu@gmail.com (Md. Rezuanul Islam)
Abstract—A high alkaline protease producing
bacterial strain was isolated and identified a local
soil sample. The organism was gram positive and
forms spore during adverse condition in the
growth medium. After various tests it was
suggested and the features agreed with the
description of Bacillus subtilis. It was also
identified as B. subtilis with 99.9% identity by API
50 CHB. The enzyme hydrolyses a number of
proteins including azocasein which suggests that
it is an extracellular alkaline protease. The
experimentally determined isoelectric point was
5.1 and the optimal enzyme activity was at 60°C
and at pH 8.5. The esterase preferentially
hydrolyzed short-chain fatty acids. Native enzyme
preparations typically showed a Michaelis
constant (Km) and Vmax of 0.40mM and 12,200 U
mg)
-1
, respectively. This microbial enzyme was
partially purified by ammonium sulfate
fractionation, dialysis, DEAE cellulose
chromatography and electrophoretic analysis.
Enzyme purity was tested by SDS-PAGE.
Quantitative estimation has shown that 40mL of
culture supernatant could dehair 2×1 cm of leather
completely in 9 hours. In future the tanneries will
use a combination of chemical and enzymatic
processes. In practical applications, protease is a
useful enzyme for promoting the hydrolysis of
proteins and showing significant industrial
applications.
Keywords—Bacillus subtilis, SDS-PAGE,
Alkaline proteases, Azocasein test, Leather
industry, Electrophoretic analysis
Introduction
Over the last few decades leather industry is based on
large scale chemicals treatment which created
worldwide environmental hazards. Leather
manufacturing is one of the industrial activities
globally wide spread, which involves the use of wide
range of chemicals many of which are hazardous,
highly toxic and obnoxiously odorous
[6, 25]
. Enzymatic
dehairing in tanneries has been envisaged as an
alternative to sulfides
[26]
. Use of enzymes for
industrial processing has received considerable
attention in recent years owing mainly to
environmental concerns
[11]
. Proteases help in
breakdown of proteins into simpler form that exist
between two amino acids of a polypeptide chain by
the process of hydrolysis
[21]
.
Leather industries are one of the most promising fields
for export to earn foreign currency in Bangladesh.
Most of the tannery industries in Bangladesh use
chemicals for dehairing that led great environmental
and health problem. Recently government of People’s
Republic of Bangladesh has taken initiative to develop
the industry from outside the city and modernize it
[18]
.
Enzymatic dehairing is suggested as an environment
friendly alternative to the conventional chemical
process
[18]
. Enzymes have been pursued as one of
the promising alternates to lime and sodium sulfide
[7]
.
Enzymes display a high capability of degrading
insoluble keratin substrates of their several potential
uses associated to the hydrolysis of keratinous
substrates and other applications
[3]
. In recent years
proteases find application in leather making among
the different industrial proteases the most widely used
enzymes in leather manufacturing
[5]
. In the back drop
of this scenario enzymes started replacing poisonous
2. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
www.jmest.org
JMESTN42350702 3371
chemicals from tannery industries. A number of
industries such as NOVO chemicals started producing
NOVOzymes for the tannery industries.
Higher cost of enzyme is one of the major factors for
the system not being practiced through found
environmentally friendly. It is essential to develop a
cost effective and eco-friendly technology by
screening for efficient enzymes from microbial
sources and producing them in large quantities by
applying recombinant DNA technology. Enzymes
found in nature are quite often not readily available in
quantities sufficient for industrial use, so use of gene
expression methods to express recombinant proteins
in suitable heterologous expression systems is
required
[2]
. Genetic engineering could be used to
increase the gene copy number as an effective
method for improving enzyme productivity
[13]
.
Secretion of recombinant proteins is a common
strategy for heterologous protein expression. The
major goal of the research showing that proteases
enzyme can be utilized in enzymatic dehairing of cow
skin in tannery industry to control the environment
from pollution, which is a prerequisite for
biotechnological applications.
Materials and Methods
Microorganism, Culture Medium and Growth
Conditions: Soil samples were collected from the
poultry wastes in Savar, after serial dilution, culture
was given in LB broth media from the sample for 16 h
at 37°C. At the next day single colony was found.
Among them few colonies were identified on the basis
of different colony morphology. Each colony was
inoculated into screw capped test tubes containing
autoclaved feather with liquid broth media and
incubated overnight at 37°C with shaking at 160 rpm.
One media was used as negative control. Chemicals
used in the experiment were from Oxoid Ltd.
(Basingstoke, UK), Merck AG (Darmstadt, Germany),
and Sigma (USA). Azokeratin was synthesized based
on the method described in a previous study
[20]
.
Isolation and Identification of Bacteria from Local
Soil Sample: The soil sample was collected from the
poultry wastes in Savar, after serial dilution, culture
were given in LB broth media from the sample for 16 h
at 37°C. At the next day single colony was found.
Among them few colonies were identified on the basis
of different colony morphology. Each colony was
inoculated into screw capped test tubes containing
autoclaved feather with liquid broth media and
incubated overnight at 37°C with shaking at 160 rpm.
One media was used as negative control. Gram’s
staining; morphological studies, physiological and
biochemical characteristics of the isolate were
investigated according to Bergey’s Manuals [24]. A
rapid bacterial identification test kit for Bacillus, API 50
CHB (BioMerieux, France), was used to identify
species of bacteria.
Biochemical & Microbiological tests for the
characterization of the organism: To identify the
biochemical properties of the organism different tests
were performed. For correct interpretation of the
results in every test Escherichia coli was taken as
control. The carbohydrate tests that were performed
are the Glucose, Lactose, Ribose, Sucrose, Mannitol,
Adonitol, Arabinose, Sorbitol, and Maltose. Others
Biochemical tests that were performed are the
Hydrogen sulfide test, Motility Test, Indole Production
Test, Citrate Utilization Test, Nitrate Reduction Test,
Oxidase test (young culture), Catalase Test, Urease
test, Indole (SIM) test, Methyl Red (MR) Voges-
Proskauer (VP) Test, Starch Hydrolysis Test and
Gelatin Liquefaction Test. Some Microbiological tests
that were performed are the Gram staining for the
Bacteria, Spore staining, colony morphology and
growth curve determination.
Production of Protease and Proteolytic Activity
Evaluation by Azocasein Test: The microorganism
was cultivated in sterile nutrient broth medium. The
culture was grown overnight on a rotary shaker at 150
rpm and incubated at 37°C for 15-20 hours. The
culture was then centrifuged at 10000 rpm for 10
minutes at 4°C. The supernatant was collected and
used as crude enzyme sample. Proteolytic activities
were assayed by Azocasein test, described by Kreger
and Lockwood (1981) was done. Here azocasein is
used as a substrate. Optical density was measured at
440 nm.
Evaluation of Growth profile and protease activity
of the organism at 37ºC: The organism was grown in
nutrient broth at 37ºC. Samples were taken at
different time interval and absorbance was taken at
600nm to measure the growth profile. The growth
profile of the organism showed that the organism
showed optimum growth after about 24 hours and the
protease activity was the maximum after 26 hours of
incubation. In the initial stage of growth there was
basal level of extracellular protease which increased
with the increase of time. The result showed that there
was differential synthesis of enzyme with growth time.
Effect of pH and temperature on enzymatic activity
and stability: For determining the effect of pH on
protease activity different buffer system with different
pH were used. Azocasein was dissolved in different
buffer solution and the enzyme assay was carried out
within pH range (4.0 to 10.5) by azocasein assay
method. All of them were used at 0.05M
concentration.
3. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
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JMESTN42350702 3372
Table-1: Different buffer used and their p
H
ranges.
Buffer PH
range
Acetate buffer 4.0-5.6
Sodium phosphate buffer 5.6-8.0
Tris HCl buffer 7.5-8.9
Glycine-NaOH buffer 8.6-10.5
For the determination of the effect of temperature, the
reaction medium was incubated at varied temperature
and the protease activity was determined. For this
purpose the enzyme preparation was added to a
mixture of 1 mg 1 % azocasein solution, 0.1 ml of 0.06
M CaCl2 and buffer (0.2 M Tris-HCI buffer, pH 8.0)
and incubated at 37°, 40°, 50°,60°,65°C temperatures.
Evaluation of Dehairing Capability of the Enzyme:
For dehairing studies, the organism was grown in
nutrient broth at 37ºC for around 20 hours. Then it
was centrifuged at 4000 rpm for 8 minutes. The cell
free supernatant was added on detergent washed
goat skin to observed enzymatic dehairing capability
of the organism. Sodium azide was used at 1% so
that no organism can grow. Nutrient broth was used
as control.
Determination of Ammonium Sulfate Fractionation
and Dialysis of Protein Mixtures: All subsequent
purification steps were carried out at 0–4 °C.
For Ammonium sulfate fraction of protein mixtures, 20
hours grown bacterial culture was centrifuged at 8000
rpm for 6 minutes. The cell free supernatant was then
saturation with ammonium sulfate slowly but
frequently to dissolve in crude culture supernatant.
After 60% saturation culture supernatant was kept in
freeze for 12 hours. After that time maximum protein
was precipitated.
Then centrifuged at 14000 rpm for 7-10 minutes to
collect the precipitates and precipitates were
redissolved in Trise-HCl (0.1M, pH 7.7) buffer.
Dialysis was carried out to remove the ammonium
salts in a cellophane bag for 8-12 hours using Trise-
HCl buffer. Then the collected sample was stored at -
20
o
C for chromatographic analysis.
Ultra Filtration by Millipore Centricons: Ultra
filtration using centricons was used to separate the
proteins having molecular weight around 100 KDa.
The protein having molecular weight 100 KDa or
above were retained in the upper part of the centricon
while small proteins were passed through the
membrane filtrate after centrifugation for 30 minutes at
5000 rpm in a Sorval super speed centrifuge.
Ion-Exchange Column Chromatography for
Protein Purification: The enzyme solution was
applied to a DEAE cellulose powder in 0.1M Trise-HCl
buffer (pH 7.5) in a beaker and left it to swell for few
hours. The gel suspension was packed in a column of
desired length. After packing the column was
equilibrated with 0.1M Trise-HCl buffer. The proteins
were eluted according to their molecular weight from
the column with same buffer by linear and the
adsorbed proteins were then eluted with a linear
gradient of 0.1–5.0 M NaCl in the same buffer at a
flow rate of 12 ml/ min). The absorbance was taken at
280nm to measure the OD of collected fractions (200
tubes). The most active fractions were concentrated
from 15ml to 3ml by PEG-6000. Then the
concentrated sample was stored at -20
o
C for gel
analysis.
Determination of Kinetic Parameters and
Isoelectric point: The kinetic parameters Km and
Vmax were determined in 30 mMTris–Cl, pH 9.0, at
25
º
C over the substrate concentration range from 0.01
to 5 mM p-nitrophenyl acetate. Analytical isoelectric
focusing of the purified enzyme was performed with
an AmpholinePAGplate precast polyacrylamide gel
(Amersham Biosciences), with pH values ranging from
3 to 10, and the broad pI calibration kit (Amersham
Biosciences) as pI marker.
Examination of Enzyme Purity: As described by
Laemmli (1970), the protein purity of the enzyme was
evaluated by SDS-PAGE using 1 mm thick slab gels
containing 14% (w/v) separating gels and 5% (w/v)
stacking gels. After running the gel was fixed
overnight in a solution of TCA and stained with
Coomassie brilliant blue G-250 using the
ultrasensitive method. Alternatively the gels were
submitted to silver staining. It was then kept immersed
in freshly prepared destaining solution till the gel
background became transparent. The electrophoretic
migration of the protein was compared with that of
low-molecular-mass protein markers (Pharmacia,
Sweden). Zymography was determined according to
the method described in the study of Riffel et al.
Results
Morphological and Biochemical Characterization
of the Isolated Soil Bacterium: The main object of
this work was to isolate and characterize thermophilic
enzyme which could specifically be used for dehairing
the hides and skins of cattle in the tannery industries.
In this connection three ways were planned. One was
to isolate thermophilic organism from different natural
sources. The others is to characterize and
identification of the isolated organism. The growth
phenotype and some of the biochemical
characteristics of the organism was determined. This
organism was characterized and identified as a
member of gram positive Bacillus sp. by several test.
The features agreed with the description of Bacillus
subtilis in Bergey’s Manual of Systematic Bacteriology
[24]
. It was also identified as B. subtilis with 99.9%
identity by API 50 CHB. So this bacteria is named
here as a Bacillus subtilis. The results are presented
here in a table-2.
Assay for Proteolytic Activity of the Enzyme:
Proteolytic activities were assayed by Azocasein test,
described by Kreger and Lockwood (1981) was done.
Here azocasein is used as a substrate. The proteolytic
activity was found as 21.13 units for the sample. One
unit of proteolytic activity is defined as the amount of
4. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
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JMESTN42350702 3373
enzyme that produces an increase in the absorbance
of 0.01 at 440nm.
Table-2: Morphological and biochemical test for the
characterization of B. subtilis.
Test performed Observations Results
Streak plate
isolation:
NA at 37°C Milky colonies Positive
Gram stain Small violate colonies
singly
Gram positive
rods
Spore stain Green color appeared Spore forms
Cultural
characteristics:
Catalase Test Bubbles formed Positive
Indole (SIM)Test Bright red ring,
growth away
Negative
Nitrate
Reduction Test
No color change after
zinc dust addition
Positive
Urease Test no bright pink color Negative
Methyle Red
Test
deep red ring formed Positive
Sucrose
Fermentation
yellow Negative
Gelatin
Hydrolysis
remain liquefied at
4ºC
Positive
Voges Proskauer
Test
weak red ring formed Positive
Starch hydrolysis bright zone Positive
Glucose Yellow color Positive for acid
and negative for
gas
Citrate test change in color Positive for
citrate utilization
Determination of Growth profile and protease
activity of the organism at 37ºC: The Bacteria was
grown in nutrient broth at 37ºC. Samples were taken
at different time interval and absorbance was taken at
600nm to measure the growth profile.
Figure-1: Graphical presentation of growth of bacteria
and protease activity at different time interval at 37º C.
The growth profile of the organism showed optimum
growth after about 24 hours and the protease activity
was the maximum after 26 hours of incubation. In the
initial stage of growth there was basal level of
extracellular protease which increased with the
increase of time. The figure-1 shows that there was
differential synthesis of enzyme with growth time.
Effect of p
H
on Protease Activity from the
Organism: The pH of the reaction media can affect
the protease activity. For this purpose the enzyme
activity over a pH range between 4 and 11 was
studied.
The enzyme shows its maximum activity at pH 8.5.
The activity decline at pH 8.0 or above 8.5. Therefore
pH 8.5 might be the optimum pH for enzyme activity.
Additionally, its optimum pH was similar to that of
previous reports
[23]
. Most proteases are active in
neutral to alkali conditions, from pH 7.0 to pH 9.5. For
example, the activity optimum of protease from
Mycobacterium kr10 is pH 7.0, B. pumilus FH9 of pH
8.0
[9]
, Fervido bacterium islandicum AW-1 of pH 9.0
[19]
.
Figure-2: Graphical presentation of effect of pH on
protease activity.
The figure-2 shows that the enzyme activity increase
with the increase of pH of the media and the optimum
pH is 8.5 for the activity of protease enzyme in Tris-
HCL buffers. The results showed that the optimum pH
of the protease enzyme was 8.5. Studies on growth
temperature and pH suggest that the organism might
be alkaline and thermophilic Bacillus
Effect of Temperature on Enzyme Activity: The
activity of the enzyme was measured over a range of
temperature (0°C, 4°C, 20°C, 30ºC, 37ºC, 40ºC, 50ºC,
60ºC, 65ºC, 80°C) and the result is presented in
figure-3
The enzyme activity is increased with the increase of
temperature. The experiment was reported 2 times
and the result is reproducible. There was a significant
increase in enzyme activity between 20°C to 55°C.
The enzyme seems to be active at 60°C and its
activity declines as the temperature increase beyond
60°c. At 80°c the enzyme has very little activity. This
suggests that the enzyme might be a thermostable
enzyme.
Figure-3 shows that the protease was active over a
temperature range of 4°C ~80°C, with an optimum at
60°C. Most proteases possess an activity optimum in
the range of 30~80°C, for example, protease from B.
0
0.5
1
1.5
2
2.5
4 6 8101213141516182022242628303234
Absorbance
Time (hours)
Growth profile
Enzyme activity
5. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
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JMESTN42350702 3374
pseudofirmusAL-89 is of 60~70°C
[10]
and a few have
exceptionally high temperature optimum of 100 °C
[19]
.
Figure-3: Graphical presentation of protease activities
at different temperature.
Evaluation Dehairing Capability of the Isolated
Protease Enzyme: The cell-free supernatants were
used as sources of crude enzyme. The treated skins
and controls showed visible differences after 9 h
incubation. No color alteration was observed, although
the presence of depilated areas was noticed in the
skins treated with enzymes. When hairs were pulled
with a forceps, they were very easily released after
enzyme treatment.
After 9 h incubation intact hairs could be taken out of
the skins easily by simple scraping. In controls, hair
loosening was not observed, even by the mechanical
action of a forceps. This result was much better than
other different bacteria that also caused dehairing.
Proteases have been used in the hide dehairing
process, where dehairing is carried out at pH values
between 8-10
[14]
. In most cases the enzymes work
and bring about efficient dehairing within 6-20h.
Figure-4: Direct dehairing activity of the enzyme-A
(Control) and B (100% Hair removed).
Comparison of dehairing ability of Bacillus
subtilis with other bacteria: Dehairing ability of the
protease produced by our strain and other bacterial
protease showed that our bacterial protease is very
fast in dehairing compared to other three.
Table-3: Comparison of dehairing ability of B. subtilis
with other bacteria (1).
Time of incubation
for dehairing
Change of
color of leather
Bacillus subtilis 9h no change
Vibrio sp kr2 24h no change
Flavobacterium
sp kr6
24h no change
Bacillus sp kr10 24h no change
Partial Purification of Protease Enzyme: To remove
unwanted proteins from the crude enzyme solution,
40–80% saturation of (NH4)2SO4 had the best effect
on enzyme purification. Most of the protein in bacterial
culture filtrate precipitated at 60% saturation. The
obtained most active enzyme protein preparation
could be obtained at the ammonium sulphate level of
60%. This result was in complete accordance with
other workers
[15]
.
Figure-5: Graphical presentation of OD of collected
fractions from DEAE cellulose column
chromatography.
The overall purification factor was about 22.6 fold and
the final yield was 51%. The final product had a
specific activity of about 839.41 U/mg. Protein
purification and different enzymetic properties of the
protease are presented in a table-4. Ion-exchange
DEAE cellulose column chromatography was for
protein purification. The desired enzyme was found in
53-55 numbers tube by Azocasein test. The result is
presented in figure-5
Figure-5 shows that the desired enzyme was found in
53-55 numbers of tubes/fractions and it was also
found that 54 numbers of tube/fraction contains large
amount of desired enzyme. Enzyme purity was tested
by SDS-PAGE according to Laemmli (1970) and
operated at 4°C. It was found that a single band is
appeared in the gel. It proves that the enzyme has
purified and separated.
BA
6. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
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JMESTN42350702 3375
Table-4: Protein purification and different enzymatic
properties of the protease.
Protein
purefication
status
Protein
conc.
mg/mL
Protease
activity
Enzyme
unites/
mL
Specific
activity
Protein
purificati
-on fold
Crude culture
supernatant
0.82 1.159 289.75 350 1
60%
saturation
with
ammonium
sulfate
3.52 1.79 447.5 127 2.3
Removal of
salt by
dialysis
1.96 0.967 242 123 2.9
Ultrafiltratio
n by
centricon
1.36 1.01 252.5 185 4.9
Gell filtration
chromatogra
phy
135µg/
mL
0.217 54.25 54.41 11.5
Kinetic Parameters and Isoelectric point: The kcat
kinetic parameter was determined using some
common acetylated substrates and the values. The
Michaelis constant Km for alkaline proteases was 0.40
€ 0.02 mM and the maximal velocity Vmax was
12,200 € 500 U mg)
-1
. The pI of the protein was
estimated by isoelectric focusing to be approximately
5.1, in agreement with the theoretically predicted pI
value of 5.0.
Discussion
Novel protease enzyme was isolated from local soil
bacterium showing remarkable dehairing activity of
cow hides and skins both qualitatively and
quantitatively. After various biochemical
characteristics, morphological tests suggested and the
features agreed with the description of B. subtilis in
Bergey’s Manual of Systematic Bacteriology
[21]
.
Azocasein assay developed by Kreger and Lockout is
a well accepted method for the assay of wide variety
of protease having overlapping specificity. The
enzyme hydrolyses a number of proteins including
Azocasein which suggest that it is an extracellular
protease
[8]
. Bacillus species have been reported to
produce proteases
[28]
.
Therefore, it may be called a very good method for the
large scale screening of bacterial protease
[12]
. In
growth curve determination showed that the growth of
the organism is increased with the increase of
incubation period and the growth reached maximum
at around 24 hours of incubation and the protease
activity was the maximum of the 26 hours culture.
The enzyme seems to have an optimum temperature
of 60ºC. Most proteases possess an activity optimum
in the range of 30~80 °C, for example, protease from
B. pseudofirmusAL-89 is of 60~70 °C
[6]
, Nocardiopsis
sp. TOA-1 is of 60 °C and a few have exceptionally
high temperature optimum of 100 °C
[19]
. The enzyme
seems to have an optimum pH of 8.5. Additionally, its
optimum pH was similar to that of previous reports
[22]
.
B. subtilis strains had been widely utilized for enzyme
production, including the proteases
[17]
.
Enzymatic dehairing may be the ideal process.
Quantitative estimation has shown that 40mL of
culture supernatant could dehair 2×1 cm of leather
completely in a 9 hours. After 9h incubation intact
hairs could be taken out of the skins easily by simple
scraping. This shows that the bacterial isolate
moderate to high amount of enzyme for dehairing.
Enzymatic dehairing in tanneries has been envisaged
as an alternative to sulfides
[20]
. A significant feature of
the enzymatic dehairing process is complete hair
removal and minimal usage of sulfide and the
decomposition products formed from the tannery
wastewater, with great improvement in wastewater
quality as a result.
A trial was given to obtain the partially purified
proteases from the culture supernatant of Bacillus sp.
from one hand to create an interesting comparative
study of the characteristics of the purified enzyme
preparations from the other hand. This microbial
enzyme was partially purified by ammonium sulphate
fractionation, dialysis, DEAE cellulose
chromatography and electrophoretic analysis. The
obtained most active enzyme protein preparation
could be obtained at the ammonium sulphate level of
60%. This result was complete accordance with other
workers
[15]
. The protease precipitated by the
ammonium sulphate had been reported in many
previous studies
[28]
. The precipitates were found to be
very active after the dialysis. This gave 2.9 fold
purification of the proteins. Ultra filtration is another
method for the separation of proteins of different
molecular weight
[26]
.Proteins having molecular weight
higher than or equal to 100kDa were used. In this
process the protein were purified to 4.9 fold.
After ultra filtration protein was further purified by gel
filtration chromatography using DEAE cellulose. This
method is very laborious and time consuming but
separation of protein is very reliable. Three different
protein picks of different molecular weight was found
and one of the pick showed considerable enzyme
activity
[4]
. In this process the protein was purified to
11.5 fold. The subunit molecular mass of the protease
was estimated by comparing the electrophoretic
mobility of the protease with the electrophoretic
mobilities of marker proteins. It was found that a
single band was appeared in the gel indicating the
enzyme has fully separated and purified. The level of
purification is higher than those reported in other
similar papers
[16].
As the bacterial protease showed
high activity in dehairing of cow skin and our next
target is to introduce it to the tannery industries, so
that they can use it instead of hazardous chemicals
for better leather quality and most importantly for a
better environment.
7. Journal of Multidisciplinary Engineering Science and Technology (JMEST)
ISSN: 3159-0040
Vol. 2 Issue 12, December - 2015
www.jmest.org
JMESTN42350702 3376
Conclusion
Bacterial alkaline protease has got its particular eco
friendly technical applications in leather processing,
detergent and feathers digestion to feed in
Bangladesh. The results showed that the B. subtilis
proteases enzyme can be utilized in enzymatic
dehairing of cow skin in tannery industry to control the
environment from pollution, which is a prerequisite for
biotechnological applications. The cultural
characteristics and biochemical tests of the organism
suggest that it is a thermophilic, Gram positive, spore
forming and aerobic bacteria. The characterization of
protease so far showed that it is an alkaline protease,
highly active at temperature near 60ºC. Finally, it
plans to clone and over-express the genes encoding
enzymes for large scale industrial production and
commercial use for pretreatment of industrial residues
from leather industry for biogas production.
Acknowledgments
Many thanks to Professor Dr. Mustafizur Rahman,
Dept. of Biochemistry, Gono University, Savar,
Bangladesh for their Financial supporting the work
and also thanks to Professor Dr. Md Rezuanul Islam,
Dept. of Biotechnology and Genetic Engineering,
Islamic University, Kushtia, Bangladesh for proper
support, guidance and help for data analysis.
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