Proteases can be classified into four main types - serine, cysteine, aspartic, and metallo proteases. Serine proteases contain a catalytic serine residue and include subtilisins. Cysteine proteases contain a catalytic cysteine-histidine dyad and include papain. Metalloproteases require a divalent metal ion like zinc and include thermolysin. The document discusses the classification, sources, and applications of various protease enzymes.
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
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
Introduction :
Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria)
The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum.
The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war.
Penicillin was the first important commercial product produced by an aerobic, submerged fermentation
Steps involved in fermentation products producing a viable product output.various steps and process were explained in them. A semester syllabus of undergraduate microbiology student in his/her semester -5 in paper -6 . I think this might be helpful to you and have a good response after reading this .thank you.
Introduction :
Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria)
The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum.
The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war.
Penicillin was the first important commercial product produced by an aerobic, submerged fermentation
Steps involved in fermentation products producing a viable product output.various steps and process were explained in them. A semester syllabus of undergraduate microbiology student in his/her semester -5 in paper -6 . I think this might be helpful to you and have a good response after reading this .thank you.
Cette étude propose d’évaluer la capacité de la combinaison d’une xylanase (x: Ronozyme® WX (CT)), d’une amylase (a: Ronozyme® A) et une protéase (p: Ronozyme® ProAct (CT)) à compenser une réduction d’énergie métabolisable et de protéine brute d’un aliment à base de blé, de maïs et de soja destiné aux poulets de chair. La combinaison des trois enzymes (xap) a été comparée.
Parlez-vous avec un expert en Twitter: @FarukMurtala
Alkaline Protease - One of the class of protease enzyme.
An extracellular enzyme.
Performs proteolysis, that is, protein catabolism by hydrolysis of the peptide bonds.
Active at alkaline pH 8 to 12 and at temperature 30⁰-80⁰C.
Molecular weight is about 20,000 to 45,000 Dalton.
The structure is determined by X-ray crystallography.
EC (Enzyme Commission) Number: 3.4.21–24.99
In 1971, Japanese scientist Koki Horikoshi first reported the production of alkaline protease from bacteria.
DNA is really long, and the nucleus of a cell is really tiny. You might think that the DNA would get tangled, and you'd be right. Here are some methods the cell uses to keep things from getting out of control.
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
Radiation. Plants Immunity. Toxicity of Plants after irradiation.Dmitri Popov
A high level of Plant Vacuolar Protease, activated after moderate and high doses of radiation, possible playing role as radiation toxin and can induce development of Acute Radiation Syndromes in mammals after ingestion.
Complex Of Enzymes Plus is recommended as a dietary supplement, an additional source of rutin, zinc and digestive enzymes like bromeline-papain-tripsin-amylase-lipase-protease etc. which have a favorable effect on the digestion process and promote complete breakdown of food and its uptake as well; thereby improving general health and well-being
based on class feedback, i've switched the presentations to a black&white template. this is easier to see in classroom presentation and most.definitely easier to print out legible notes!
this ppt covers about amino acids, classification, protein ,classifications, structure, denaturation, structure & fnctional relationship with applied aspects.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
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.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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 .
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
(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.
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.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
3. Introduction
Proteases are the single class of enzymes which occupy
a pivotal position with respect to their applications in
both physiological and commercial fields.
Proteolytic enzymes catalyze the cleavage of peptide
bonds in other proteins. Proteases are degradative
enzymes which catalyze the total hydrolysis of proteins.
4. Introduction
proteases conduct highly specific and selective
modifications of proteins such as:
Activation of zymogenic forms of enzymes by limited
proteolysis,
Blood clotting and lysis of fibrin clots,
Processing and transport of secretory proteins across
the membranes.
5. Introduction
The current estimated value of the worldwide sales of
industrial enzymes is $1 billion.
Of the industrial enzymes, 75% are hydrolytic.
Proteases represent one of the three largest groups of
industrial enzymes and account for about 60% of the total
worldwide sale of enzymes (Fig. 1).
10. Sources Of Proteases
Plant Proteases
Animal Proteases
Microbial Proteases
Papain,
Bromelain,
Keratinases,
Ficin
Such as
11. Sources Of Proteases
Plant Proteases
Animal Proteases
Microbial Proteases
Trypsin,
Chymotrypsin,
Pepsin,
Rennins
Such as
12. Sources Of Proteases
Plant Proteases
Animal Proteases
Microbial Proteases Bacterial,
Fungal,
Viral
Such as Neutral and Alkaline Proteases
13. Sources Of Proteases
Plant Proteases
Animal Proteases
Microbial Proteases Bacterial,
Fungal,
Viral
Such as
Acid, Neutral and Alkaline
Proteases
14. Sources Of Proteases
Plant Proteases
Animal Proteases
Microbial Proteases Bacterial,
Fungal,
Viral
Such as
Serine, Aspartic, and Cysteine
Peptidases (All are EndoPeptidases,
there are no metallopeptidases)
17. Classification Of Proteases
Exopeptidases
Endopeptidases
Aminopeptidases,
Carboxypeptidases,
Omega peptidases
Dipeptidyl Peptidases
Tripeptidyl Peptidases
Aminopeptidase I from Escherichia coli is a large protease (400,000 Da).
It has a broad pH optimum of 7.5 to 10.5 and requires Mg2+ or Mn2+.
The Bacillus licheniformis aminopeptidase has a molecular weight of 34,000.
It contains 1 g-atom of Zn2+ per mol, and its activity is enhanced by Co2+ ions.
Aminopeptidase II from B. stearothermophilus is a dimer with a molecular
weight of 80,000 to 100,000 and is activated by Zn2+, Mn2+, or Co2+ ions.
18. Classification Of Proteases
Exopeptidases
Endopeptidases
Aminopeptidases,
Carboxypeptidases,
Omega peptidases
Serine type protease
Metalloprotease
Cysteine type protease
Peptidyl dipeptidase
Dipeptidases
Carboxypeptidases can be divided into three major groups, serine carboxypeptidases,
metallocarboxypeptidases, and cysteine carboxypeptidases, based on the nature of the amino acid
residues at the active site of the enzymes.
The serine carboxypeptidases isolated from Penicillium spp., Saccharomyces spp., and Aspergillus spp.
are similar in their substrate specificities but differ slightly in other properties such as pH optimum,
stability, molecular weight, and effect of inhibitors.
Metallocarboxypeptidases from Saccharomyces spp. and Pseudomonas spp. require Zn2+ or Co2+ for
their activity.
The enzymes can also hydrolyze the peptides in which the peptidyl group is replaced by a pteroyl
moiety or by acyl groups.
19. Classification Of Proteases
Exopeptidases
Endopeptidases Serine protease
Cysteine protease
Aspartic protease
Metalloprotease
S
C
A
M
And U for Unknown types
21. Classification Of Proteases
Serine Proteases
Presence of a serine group in their active site.
Are found in the exopeptidase, endopeptidase, oligopeptidase, and omega peptidase groups.
Based on their structural similarities, serine proteases have been grouped into 20 families, which
have been further subdivided into about six clans with common ancestors.
The primary structures of the members of four clans, chymotrypsin (SA), subtilisin (SB),
carboxypeptidase C (SC), and Escherichia D-Ala–D-Ala peptidase A (SE) are totally unrelated,
suggesting that there are at least four separate evolutionary origins for serine proteases.
Clans SA, SB, and SC have a common reaction mechanism consisting of a common catalytic triad of
the three amino acids, serine (nucleophile), aspartate (electrophile), and histidine (base).
Another interesting feature of the serine proteases is the conservation of glycine residues in the
vicinity of the catalytic serine residue to form the motif Gly-Xaa-Ser-Yaa-Gly.
22. Classification Of Proteases
Serine Proteases
Serine proteases are recognized by their irreversible inhibition by 3,4-
dichloroisocoumarin (3,4-DCI), L-3-carboxytrans 2,3-epoxypropyl-leucylamido (4-
guanidine) butane (E.64), diisopropylfluorophosphate (DFP), phenylmethylsulfonyl
fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK).
Serine proteases are generally active at neutral and alkaline pH, with an optimum
between pH 7 and 11.
They have broad substrate specificities including esterolytic and amidase activity.
Their molecular masses range between 18 and 35 kDa, for the serine protease from
Blakeslea trispora, which has a molecular mass of 126 kDa.
The isoelectric points of serine proteases are generally between pH 4 and 6.
Serine alkaline proteases that are active at highly alkaline pH represent the largest
subgroup of serine proteases.
23. Classification Of Proteases
Serine Proteases
(i) Serine alkaline proteases.
They are produced by several bacteria, molds, yeasts, and fungi.
They are inhibited by DFP or a potato protease inhibitor but not by tosyl-L-phenylalanine
chloromethyl ketone (TPCK) or TLCK.
They hydrolyze a peptide bond which has tyrosine, phenylalanine, or leucine at the
carboxyl side of the splitting bond.
The optimal pH of alkaline proteases is around pH 10, and their isoelectric point is around
pH 9.
Their molecular masses are in the range of 15 to 30 kDa.
Although alkaline serine proteases are produced by several bacteria such as Arthrobacter,
Streptomyces, and Flavobacterium spp., subtilisins produced by Bacillus spp. are the best
known.
Alkaline proteases are also produced by S. cerevisiae and filamentous fungi such as
Conidiobolus spp. and Aspergillus and Neurospora spp.
24. Classification Of Proteases
Serine Proteases
(ii) Subtilisins.
Two different types of alkaline proteases, subtilisin Carlsberg and subtilisin Novo or bacterial
protease Nagase (BPN9), have been identified.
Subtilisin Carlsberg produced by Bacillus licheniformis was discovered in 1947 by Linderstrom, Lang,
and Ottesen at the Carlsberg laboratory.
Subtilisin Novo or BPN9 is produced by Bacillus amyloliquefaciens.
Subtilisin Carlsberg is widely used in detergents. Its annual production amounts to about 500 tons
of pure enzyme protein.
Both subtilisins have a molecular mass of 27.5 kDa but differ from each other by 58 amino acids.
They have similar properties such as an optimal temperature of 60°C and an optimal pH of 10.
They have an active-site triad made up of Ser221, His64 and Asp32.
The active-site conformation of subtilisins is similar to that of trypsin and chymotrypsin despite the
dissimilarity in their overall molecular arrangements.
25. Classification Of Proteases
Cysteine/thiol proteases.
Cysteine proteases occur in both prokaryotes and eukaryotes.
About 20 families of cysteine proteases have been recognized.
The activity of all cysteine proteases depends on a catalytic dyad consisting of
cysteine and histidine.
The order of Cys and His (Cys-His or His-Cys) residues differs among the families.
Generally, cysteine proteases are active only in the presence of reducing agents
such as HCN or cysteine.
Based on their side chain specificity, they are broadly divided into four groups:
i. papain-like,
ii. trypsin-like with preference for cleavage at the arginine residue,
iii. specific to glutamic acid,
iv. others.
26. Classification Of Proteases
Cysteine/thiol proteases.
Papain is the best-known cysteine protease.
Cysteine proteases have neutral pH optima, although a few of them, e.g., lysosomal
proteases, are maximally active at acidic pH.
They are susceptible to sulfhydryl agents such as PCMB but are unaffected by DFP
and metal-chelating agents.
Clostripain, produced by the anaerobic bacterium Clostridium histolyticum,
exhibits a stringent specificity for arginyl residues at the carboxyl side of the
splitting bond and differs from papain in its obligate requirement for calcium.
Streptopain, the cysteine protease produced by Streptococcus spp., shows a
broader specificity, including oxidized insulin B chain and other synthetic
substrates.
Clostripain has an isoelectric point of pH 4.9 and a molecular mass of 50 kDa,
whereas the isoelectric point and molecular mass of streptopain are pH 8.4 and 32
kDa, respectively.
27. Classification Of Proteases
Metalloproteases.
Metalloproteases are the most diverse of the catalytic types of proteases.
They are characterized by the requirement for a divalent metal ion for their
activity.
They include enzymes from a variety of origins such as collagenases from
higher organisms, hemorrhagic toxins from snake venoms, and
thermolysin from bacteria.
About 30 families of metalloproteases have been recognized, of which 17
contain only endopeptidases, 12 contain only exopeptidases, and 1 (M3)
contains both endo- and exopeptidases.
Families of metalloproteases have been grouped into different clans based
on the nature of the amino acid that completes the metal-binding site; e.g.,
clan MA has the sequence HEXXH-E and clan MB corresponds to the motif
HEXXH-H.
28. Classification Of Proteases
Metalloproteases.
Based on the specificity of their action,
metalloproteases can be divided into four groups,
i. Neutral,
ii. Alkaline,
iii. Yxobacter I,
iv. Myxobacter II
All of them are inhibited by chelating agents such as
EDTA but not by sulfhydryl agents or DFP.
The neutral proteases show specificity for
hydrophobic amino acids, while the alkaline
proteases possess a very broad specificity.
Myxobacter protease I is specific for small amino acid
residues on either side of the cleavage bond, whereas
protease II is specific for lysine residue on the amino side
of the peptide bond.
29. Classification Of Proteases
Metalloproteases.
Thermolysin, a neutral protease, is the most thoroughly characterized
member of clan MA. Histidine residues from the HEXXH motif serve as Zn
ligands, and Glu has a catalytic function.
It produced by B. stearothermophilus is a single peptide without disulfide
bridges and has a molecular mass of 34 kDa.
It contains an essential Zn atom embedded in a cleft formed between two
folded lobes of the protein and four Ca atoms which impart thermostability
to the protein.
Thermolysin is a very stable protease, with a half-life of 1 h at 80°C.
30. Classification Of Proteases
Metalloproteases.
Collagenase, another important metalloprotease, was first discovered
in the broth of the anaerobic bacterium Clostridium hystolyticum as
a component of toxic products. Later, it was found to be produced by
the aerobic bacterium Achromobacter iophagus and other
microorganisms including fungi.
The action of collagenase is very specific; i.e., it acts only on
collagen and gelatin and not on any of the other usual protein
substrates.
Elastase produced by Pseudomonas aeruginosa is another important
member of the neutral metalloprotease family.
31. Classification Of Proteases
Metalloproteases.
The alkaline metalloproteases produced by Pseudomonas
aeruginosa and Serratia spp. are active in the pH range from 7 to 9
and have molecular masses in the region of 48 to 60 kDa.
Myxobacter protease I has a pH optimum of 9.0 and a molecular
mass of 14 kDa and can lyse cell walls of Arthrobacter crystellopoites,
whereas protease II cannot lyse the bacterial cells.
Matrix metalloproteases play a prominent role in the degradation of
the extracellular matrix during tissue morphogenesis, differentiation,
and wound healing and may be useful in the treatment of diseases
such as cancer and arthritis.
32. Classification Of Proteases
In summary,
proteases are broadly classified as endo- or exoenzymes on
the basis of their site of action on protein substrates.
They are further categorized as serine proteases, aspartic
proteases, cysteine proteases, or metalloproteases
depending on their catalytic mechanism.
They are also classified into different families and clans
depending on their amino acid sequences and evolutionary
relationships.
Based on the pH of their optimal activity, they are also
referred to as acidic, neutral, or alkaline proteases.
33. Mechanisem of Action of Proteases
The catalytic site of proteases is flanked on one or both
sides by specificity subsites, each able to accommodate
the side chain of a single amino acid residue from the
substrate.
These sites are numbered from the catalytic site S1
through Sn toward the N terminus of the structure and Sl’
through Sn’ toward the C terminus.
The residues which they accommodate from the substrate
are numbered Pl through Pn and P1’ through Pn’,
respectively (Fig. 2).
34. Mechanisem of Action of Proteases
S3 S2 S1 S’1 S’2 S’1
P3 P2 P1 P’1 P’2 P’1
Sn S’n
Pn P’n
Protease: N
C
C
Substrate: N
*
Fig 2: Active sites of proteases.
The catalytic site of proteases is indicated by and the scissile
bond is indicated by ; S1 through Sn and S1’ through Sn’ are
the specificity subsites on the enzyme, while P1 through Pn and
P1’ through Pn’ are the residues on the substrate
accommodated by the subsites on the enzyme.
*
35. Mechanisem of Action of Proteases
Serine Proteases:
Serine proteases usually follow a two-step reaction for hydrolysis in
which a covalently linked enzyme-peptide intermediate is formed with
the loss of the amino acid or peptide fragment.
This acylation step is followed by a diacylation process which occurs by
a nucleophilic attack on the intermediate by water, resulting in
hydrolysis of the peptide.
Serine endopeptidases can be classified into three groups based
mainly on their primary substrate preference:
(i) Trypsin-like, which cleave after positively charged residues;
(ii) Chymotrypsin-like, which cleave after large hydrophobic residues;
(iii) Elastase-like, which cleave after small hydrophobic residues.
36. Mechanisem of Action of Proteases
Serine Proteases:
The P1 residue exclusively dictates the site of peptide bond
cleavage.
The primary specificity is affected only by the P1 residues; the
residues at other positions affect the rate of cleavage.
Some of the serine peptidases from Achromobacter spp. are
lysine-specific enzymes, whereas those from Clostridium spp.
are arginine specific (clostripain) and those from
Flavobacterium spp. are post proline-specific.
Endopeptidases that are specific to glutamic acid and aspartic
acid residues have also been found in B.licheniformis and S.
aureus.
37. Mechanisem of Action of Proteases
Serine Proteases:
The chymotrypsin-like enzymes are confined almost entirely to animals, the exceptions being
trypsin-like enzymes from actinomycetes and Saccharopolyspora spp. and from the fungus
Fusarium oxysporum.
A few of the serine proteases belonging to the subtilisin family show a catalytic triad composed of
the same residues as in the chymotrypsin family; however, the residues occur in a different order
(Asp-His-Ser). Some members of the subtilisin family from the yeasts Tritirachium and Metarhizium
spp. Require thiol for their activity. The thiol dependance is attributable to Cys173 near the active-
site histidine.
The carboxypeptidases are unusual among the serine-dependent enzymes in that they are
maximally active at acidic pH.
These enzymes are known to possess a Glu residue preceding the catalytic Ser, which is believed to
be responsible for their acidic pH optimum. Although the majority of the serine proteases contain
the catalytic triad Ser-His-Asp, a few use the Ser-base catalytic dyad. The Glu-specific proteases
display a pronounced preference for Glu-Xaa bonds over Asp-Xaa bonds.
38. Mechanisem of Action of Proteases
Aspartic Proteases:
Aspartic endopeptidases depend on the aspartic acid residues for
their catalytic activity.
A general base catalytic mechanism has been proposed for the
hydrolysis of proteins by aspartic proteases such as penicillopepsin
and endothiapepsin.
Crystallographic studies have shown that the enzymes of the pepsin
family are bilobed molecules with the active-site cleft located between
the lobes and each lobe contributing one of the pair of aspartic acid
residues that is essential for the catalytic activity.
The lobes are homologous to one another, having arisen by gene
duplication.
39. Mechanisem of Action of Proteases
Aspartic Proteases:
The retropepsin molecule has only one lobe, which carries only one aspartic residue, and the
activity requires the formation of a noncovalent homodimer.
In most of the enzymes from the pepsin family, the catalytic Asp residues are contained in an Asp-
Thr-Gly-Xaa motif in both the N- and C-terminal lobes of the enzyme, where Xaa is Ser or Thr,
whose side chains can hydrogen bond to Asp. However, Xaa is Ala in most of the retropepsins. A
marked conservation of cysteine residue is also evident in aspartic proteases. The pepsins and the
majority of other members of the family show specificity for the cleavage of bonds in peptides of
at least six residues with hydrophobic amino acids in both the Pl and Pl9 positions.
The specificity of the catalysis has been explained on the basis of available crystal structures.
The structural and kinetic studies also have suggested that the mechanism involves general acid-
base catalysis with lytic water molecule that directly participates in the reaction (Fig. 3A).
This is supported by the crystal structures of various aspartic protease-inhibitor complexes and by
the thiol inhibitors mimicking a tetrahedral intermediate formed after the attack by the lytic water
molecule.
40. Mechanisem of Action of Proteases
Metalloproteases:
The mechanism of action of metalloproteases is slightly different from that of
the above-described proteases.
These enzymes depend on the presence of bound divalent cations and can be
inactivated by dialysis or by the addition of chelating agents.
For thermolysin, based on the X-ray studies of the complex with a hydroxamic
acid inhibitor, it has been proposed that Glu143 assists the nucleophilic attack
of a water molecule on the carbonyl carbon of the scissile peptide bond, which
is polarized by the Zn2+ ion.
Most of the metalloproteases are enzymes containing the His-Glu-Xaa-Xaa-His
(HEXXH) motif, which has been shown by X-ray crystallography to form a part
of the site for binding of the metal, usually zinc.
41. Mechanisem of Action of Proteases
Cysteine proteases:
Cysteine Proteases catalyze the hydrolysis of carboxylic acid derivatives through a double-
displacement pathway involving general acid-base formation and hydrolysis of an acyl-thiol
intermediate.
The mechanism of action of cysteine proteases is thus very similar to that of serine proteases.
A striking similarity is also observed in the reaction mechanism for several peptidases of different
evolutionary origins.
The plant peptidase papain can be considered the archetype of cysteine peptidases and
constitutes a good model for this family of enzymes.
They catalyze the hydrolysis of peptide, amide ester, thiol ester, and thiono ester bonds.
The initial step in the catalytic process (Fig. 3B) involves the noncovalent binding of the free
enzyme (structure a) and the substrate to form the complex (structure b).
This is followed by the acylation of the enzyme (structure c), with the formation and release of
the first product, the amine R’-NH2. In the next deacylation step, the acyl-enzyme reacts with a
water molecule to release the second product, with the regeneration of free enzyme.
42. Mechanisem of Action of Proteases
Cysteine proteases:
The enzyme papain consists of a single protein chain folded to form two domains
containing a cleft for the substrate to bind.
The crystal structure of papain confirmed the Cys25-His159 pairing.
The presence of a conserved aspargine residue (Asn175) in the proximity of catalytic
histidine (His159) creating a Cys-His-Asn triad in cysteine peptidases is considered
analogous to the Ser-His-Asp arrangement found in serine proteases.
Studies of the mechanism of action of proteases have revealed that they exhibit different
types of mechanism based on their active-site configuration.
The serine proteases contain a Ser-His-Asp catalytic triad, and the hydrolysis of the peptide
bond involves an acylation step followed by a deacylation step.
Aspartic proteases are characterized by an Asp-Thr-Gly motif in their active site and by an
acid-base catalysis as their mechanisms of action.
The activity of metalloproteases depends on the binding of a divalent metal ion to a His-Glu-
Xaa-Xaa-His motif.
Cysteine proteases adopt a hydrolysis mechanism involving a general acid-base formation
followed by hydrolysis of an acyl-thiol intermediate.
43. Mechanisem of Action of Proteases
Studies of the mechanism of action of proteases have revealed that
they exhibit different types of mechanism based on their active-site
configuration.
The serine proteases contain a Ser-His-Asp catalytic triad, and the
hydrolysis of the peptide bond involves an acylation step followed
by a deacylation step.
Aspartic proteases are characterized by an Asp-Thr-Gly motif in
their active site and by an acid-base catalysis as their mechanisms
of action.
The activity of metalloproteases depends on the binding of a
divalent metal ion to a His-Glu-Xaa-Xaa-His motif.
Cysteine proteases adopt a hydrolysis mechanism involving a
general acid-base formation followed by hydrolysis of an acyl-
thiol intermediate.
44. Mechanisem of Action of Proteases
Fig 3: Mechanism of action of proteases.
(A) Aspartic proteases.
(B) Cysteine proteases.
Im and 1HIm refer to the imidazole and
protonated imidazole, respectively.
45. Mechanisem of Action of Proteases
Fig 3: Mechanism of action of proteases.
(A) Aspartic proteases.
(B) Cysteine proteases.
Im and 1HIm refer to the imidazole and
protonated imidazole, respectively.
46. Physiological Functions of Proteases
Proteases play a critical role in many physiological and pathological
processes such as protein catabolism, blood coagulation, cell growth
and migration, tissue arrangement, morphogenesis in development,
inflammation, tumor growth and metastasis, activation of zymogens,
release of hormones and pharmacologically active peptides from
precursor proteins, and transport of secretory proteins across
membranes.
Protein turnover
Sporulation and Conidial Discharge
Germination
Enzyme Modification
Nutrition
Regulation of Gene Expression
47. Applications of Proteases
Proteases have a large variety of applications, mainly in the detergent and food
industries.
In view of the recent trend of developing environmentally friendly technologies,
proteases are envisaged to have extensive applications in leather treatment
and in several bioremediation processes.
The worldwide requirement for enzymes for individual applications varies
considerably.
Proteases are used extensively in the pharmaceutical industry for preparation of
medicines such as ointments for debridement of wounds, etc.
Proteases that are used in the food and detergent industries are prepared in
bulk quantities and used as crude preparations, whereas those that are used in
medicine are produced in small amounts but require extensive purification
before they can be used.
48. Applications of Proteases
Detergents:
Proteases are one of the standard ingredients of all kinds of detergents ranging from those used
for household laundering to reagents used for cleaning contact lenses or dentures.
The use of proteases in laundry detergents accounts for approximately 25% of the total
worldwide sales of enzymes.
The preparation of the first enzymatic detergent, “Burnus,” dates back to 1913; it consisted of
sodium carbonate and a crude pancreatic extract. The first detergent containing the bacterial
enzyme was introduced in 1956 under the trade name BIO-40.
In 1960, Novo Industry A/S introduced alcalase, produced by Bacillus licheniformis; its
commercial name was BIOTEX.
This was followed by Maxatase, a detergent made by Gist-Brocades.
The biggest market for detergents is in the laundry industry, amounting to a worldwide production
of 13 billion tons per year.
The ideal detergent protease should possess broad substrate specificity to facilitate the
removal of a large variety of stains due to food, blood, and other body secretions.
49. Applications of Proteases
Detergents:
Activity and stability at high pH and temperature and compatibility with other chelating and oxidizing agents
added to the detergents are among the major prerequisites for the use of proteases in detergents.
The key parameter for the best performance of a protease in a detergent is its pI. It is known that a protease is
most suitable for this application if its pI coincides with the pH of the detergent solution.
Esperase and Savinase T (Novo Industry), produced by alkalophilic Bacillus spp., are two commercial
preparations with very high isoelectric points (pI 11).
A combination of lipase, amylase, and cellulose is expected to enhance the performance of protease in
laundry detergents.
All detergent proteases currently used in the market are serine proteases produced by Bacillus strains.
An alkaline protease from Conidiobolus coronatus was found to be compatible with commercial detergents
used in India and retained 43% of its activity at 50°C for 50 min in the presence of Ca2+ (25 mM) and glycine
(1 M).
50. Applications of Proteases
Leather Industries:
Leather processing involves several steps such as soaking, dehairing, bating, and tanning.
The major building blocks of skin and hair are proteinaceous.
The conventional methods of leather processing involve hazardous chemicals such as sodium sulfide, which
create problems of pollution and effluent disposal.
The use of enzymes as alternatives to chemicals has proved successful in improving leather quality and in
reducing environmental pollution.
Proteases are used for selective hydrolysis of noncollagenous constituents of the skin and for removal of
nonfibrillar proteins such as albumins and globulins.
Microbial alkaline proteases are used to ensure faster absorption of water and to reduce the time required
for soaking.
51. Applications of Proteases
Leather Industries:
Alkaline proteases with hydrated lime and sodium chloride are used for dehairing, resulting in a significant
reduction in the amount of wastewater generated.
Earlier methods of bating were based on the use of animal feces as the source of proteases; these methods
were unpleasant and unreliable and were replaced by methods involving pancreatic trypsin.
Currently, trypsin is used in combination with other Bacillus and Aspergillus proteases for bating.
The selection of the enzyme depends on its specificity for matrix proteins such as elastin and keratin, and the
amount of enzyme needed depends on the type of leather (soft or hard) to be produced.
Increased usage of enzymes for dehairing and bating not only prevents pollution problems but also is
effective in saving energy.
Novo Nordisk manufactures three different proteases, Aquaderm, NUE, and Pyrase, for use in soaking,
dehairing, and bating, respectively.
52. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
53. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
54. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
55. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
56. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
57. Applications of Proteases
Food Industry:
The use of proteases in the food industry dates back to antiquity.
They have been routinely used for various purposes such as cheesemaking, baking, preparation of soya
hydrolysates, and meat tenderization.
Dairy industry
Baking industry
Manufacture of soy products
Debittering of protein hydrolysates
Synthesis of aspartame
58. Applications of Proteases
Pharmaceutical Industry:
The wide diversity and specificity of proteases are used to great advantage in
developing effective therapeutic agents.
Oral administration of proteases from Aspergillus oryzae (Luizym and Nortase) has
been used as a digestive aid to correct certain lytic enzyme deficiency syndromes.
An asparginase isolated from E. coli is used to eliminate aspargine from the
bloodstream in the various forms of lymphocytic leukemia.
Alkaline protease from Conidiobolus coronatus was found to be able to replace
trypsin in animal cell cultures.
59. Applications of Proteases
Other Applications:
Besides their industrial and medicinal applications, proteases play an important role in
basic research.
Their selective peptide bond cleavage is used in the elucidation of structure-function
relationship, in the synthesis of peptides, and in the sequencing of proteins.
In essence, the wide specificity of the hydrolytic action of proteases finds an extensive
application in the food, detergent, leather, and pharmaceutical industries, as well as in
the structural elucidation of proteins, whereas their synthetic capacities are used for the
synthesis of proteins.