This document summarizes a study analyzing the proteases in malted and germinating barley grains. The goals were to characterize protease activity, identify individual proteases, and investigate their significance in malting. Four classes of proteases were found to be important: serine, cysteine, metallo, and aspartate. Cysteine proteases were the most active, followed by aspartate, serine, and metalloproteases. Physiological studies showed cysteine proteases activate limit dextrinase, while serine and aspartate proteases regulate alpha-amylase levels and serine proteases degrade beta-amylase. Further work will investigate the roles of aspartate and serine proteases in gibber
This presentation gives you the overall information of how enzymes are used in dairy industry and detailed explanation on production of cheese. Refer to the references for more detailed information.
Enzymes are a biological substance that accelerates the rate of various biochemical reactions in a living organism without being used up in the reaction. Their role in food processing has also been recognized for many centuries. Even before this knowledge about enzymes, they have been used in a number of processes such as the tenderization of meat using papaya leaves, soy sauce preparation, curd or cheese making, baking, brewing, etc. From animals to plants to microbial sources, enzymes may be extracted from any living organisms. Of the hundred or so enzymes being used in industries, more than half are of microbial origin. In the food industry, microbial enzymes have been extensively used to increase the diversity, variety, and quality of food. Microorganisms as an enzyme source are always preferred over other sources as large amounts of enzymes can be produced from them in a controlled manner that is also faster and cheaper. Moreover, the minimum of potentially harmful content is present in microbial enzymes in comparison to those of plants and animals. This chapter includes microbial enzymes used in food processing and the food industry, their physicochemical and biological properties, recent developments, and future prospects.
This presentation gives you the overall information of how enzymes are used in dairy industry and detailed explanation on production of cheese. Refer to the references for more detailed information.
Enzymes are a biological substance that accelerates the rate of various biochemical reactions in a living organism without being used up in the reaction. Their role in food processing has also been recognized for many centuries. Even before this knowledge about enzymes, they have been used in a number of processes such as the tenderization of meat using papaya leaves, soy sauce preparation, curd or cheese making, baking, brewing, etc. From animals to plants to microbial sources, enzymes may be extracted from any living organisms. Of the hundred or so enzymes being used in industries, more than half are of microbial origin. In the food industry, microbial enzymes have been extensively used to increase the diversity, variety, and quality of food. Microorganisms as an enzyme source are always preferred over other sources as large amounts of enzymes can be produced from them in a controlled manner that is also faster and cheaper. Moreover, the minimum of potentially harmful content is present in microbial enzymes in comparison to those of plants and animals. This chapter includes microbial enzymes used in food processing and the food industry, their physicochemical and biological properties, recent developments, and future prospects.
Role of immobilized Enzymes in Food industryJasmineJuliet
Immobilization techniques, Immobilization techniques in food industry, Immobilized Enzymes, Need for immobilization, Role of immobilized Enzymes in Food Industry, Methods of immobilization, Production of lactose free milk, Production of High Fructose corn syrups, Production of Juice in industry level by Immobilized enzymes of Pectinase, Meat tenderization by immobilized Enzymes, Immobilized Amino acylase, immobilized glucose isomerase, immobilized pectinase, Immobilized alkaline phosphatase.
In this context, there is a need to use “biodetergent or biocleaners”, which offer a better option to the synthetic detergents with respect to their biodegradability, low toxicity, non-corrosiveness environmental-friendliness, enhanced cleaning properties and their increased efficiency and stability in different formulations.
To counter these limitations, enzyme-based detergents are fast emerging as an alternative to synthetic detergents owing to their
biodegradability,
low toxicity,
non- corrosiveness,
environmental friendliness,
enhanced cleaning properties,
increased efficiency and stability in different formulations.
They are therefore also being referred to as “green chemicals”
Presently, proteases, amylases, lipases and cellulases make up the major portion of the market for industrial enzymes in cleaning applications.
Protease enzymes were first hydrolases introduced into detergent formulations specifically for the degradation of protein-based stains. Proteases have been classified according to the nucleophile or reactive component found at their catalytic sites
Changes in flour quality are and will continue to be a problem for the bakery industry. Large amounts of grain are processed by the milling industry and many resources used to secure the flour produced have a consistent quality.
The integration of enzymes in food and feed processes is a well-established approach; however there are clear evidences that dedicated research efforts are consistently being made to make the applications of biological agents more effective as well as diversified.
Various techniques have been employed such as rDNA technology and protein engineering (site-directed mutagenesis and random mutation) for the design of new/improved biocatalysts
Advances in molecular biology, evolution- ary protein engineering expertise, the (bio) computational tools, and the implementation of high-throughput meth- odologies enabling the efficient and timely screening/ characterization of the biocatalysts.
There needs to be continue efforts in the direction to have more diverse, versatile and robust enzymes to be applied in food technology
Native peptides can be regarded as surrogate markers for protease activity in biological samples. Analysis of peptides by peptidomics allows to monitor protease activity in vivo and to describe the influence of protease inhibition. To elucidate the potential of peptides as markers for in vivo protease inhibition we analyzed plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX or the dipeptidylpeptidase IV inhibitor AB192. Signals correlating with the treatment were subsequently identified and assessed with respect to protease-dependent consensus cleavage motifs and occurrence of downstream targets. It could be shown that regulated peptides were either substrates, products or downstream targets of the inhibited protease. The results from the present study demonstrate that the in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor related effects in vivo and that this method may pave the way to develop predictive biomarkers.
Role of immobilized Enzymes in Food industryJasmineJuliet
Immobilization techniques, Immobilization techniques in food industry, Immobilized Enzymes, Need for immobilization, Role of immobilized Enzymes in Food Industry, Methods of immobilization, Production of lactose free milk, Production of High Fructose corn syrups, Production of Juice in industry level by Immobilized enzymes of Pectinase, Meat tenderization by immobilized Enzymes, Immobilized Amino acylase, immobilized glucose isomerase, immobilized pectinase, Immobilized alkaline phosphatase.
In this context, there is a need to use “biodetergent or biocleaners”, which offer a better option to the synthetic detergents with respect to their biodegradability, low toxicity, non-corrosiveness environmental-friendliness, enhanced cleaning properties and their increased efficiency and stability in different formulations.
To counter these limitations, enzyme-based detergents are fast emerging as an alternative to synthetic detergents owing to their
biodegradability,
low toxicity,
non- corrosiveness,
environmental friendliness,
enhanced cleaning properties,
increased efficiency and stability in different formulations.
They are therefore also being referred to as “green chemicals”
Presently, proteases, amylases, lipases and cellulases make up the major portion of the market for industrial enzymes in cleaning applications.
Protease enzymes were first hydrolases introduced into detergent formulations specifically for the degradation of protein-based stains. Proteases have been classified according to the nucleophile or reactive component found at their catalytic sites
Changes in flour quality are and will continue to be a problem for the bakery industry. Large amounts of grain are processed by the milling industry and many resources used to secure the flour produced have a consistent quality.
The integration of enzymes in food and feed processes is a well-established approach; however there are clear evidences that dedicated research efforts are consistently being made to make the applications of biological agents more effective as well as diversified.
Various techniques have been employed such as rDNA technology and protein engineering (site-directed mutagenesis and random mutation) for the design of new/improved biocatalysts
Advances in molecular biology, evolution- ary protein engineering expertise, the (bio) computational tools, and the implementation of high-throughput meth- odologies enabling the efficient and timely screening/ characterization of the biocatalysts.
There needs to be continue efforts in the direction to have more diverse, versatile and robust enzymes to be applied in food technology
Native peptides can be regarded as surrogate markers for protease activity in biological samples. Analysis of peptides by peptidomics allows to monitor protease activity in vivo and to describe the influence of protease inhibition. To elucidate the potential of peptides as markers for in vivo protease inhibition we analyzed plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX or the dipeptidylpeptidase IV inhibitor AB192. Signals correlating with the treatment were subsequently identified and assessed with respect to protease-dependent consensus cleavage motifs and occurrence of downstream targets. It could be shown that regulated peptides were either substrates, products or downstream targets of the inhibited protease. The results from the present study demonstrate that the in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor related effects in vivo and that this method may pave the way to develop predictive biomarkers.
Large family of proteolytic enzymes
All have serine residue at their active site which plays a crucial part in the enzymatic activity.
All cleave peptide bonds, by a similar mechanism of action. They differ in their specificity and regulation.
Serine proteases include:
the pancreatic proteases: trypsin, chymotrypsin and elastase,
various tissue/intracellular proteases such as leukocyte elastase
enzymes of the blood clotting cascade
some enzymes of complement system
Many serine proteases are synthesized as inactive precursors (zymogens) which are activated by proteolysis
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
Production of enzyme Proteases: Biotechnology, pharmacyLokesh Patil
Proteases are the enzymes formed by mixture of proteinases and peptidases which provides platform for hydrolysis of proteins. About 500 tons of proteases are produced per year making protease 2nd largest producing enzyme.
Alkaline serine proteases are specifically manufactured on larger scale for production of detergents, as it have maximum stability in highly basic conditions and they are stable even at high temperature. Also they have stability towards the complexation, they do net directly get inhibited by the EDTA (Ethylene Diamino Tetra Acetic acid). proteases can be inhibited by the DFP (Di Isopropyl Flurophosphate)
Production of Enzyme - Lipase.
INTRODUCTION: Lipases are hydrolases capable of catalyzing the hydrolysis of Triglycerols (TAGs) into Glycerol and Fatty acids (FAs).
These enzymes operate at the interfaces of Biphasic systems, which is a phenomenon known as interfacial activation.
These do not require co-factors and are easily immobilized on different matrices.
The active sites of lipases are generally characterized by amino acid triad composed of serine, histidine and aspartate.
Lipases exihibit region-selective properties and enantioselective catalytic behaviour and are considered to be the most versatile catalyst in lipid biotechnology.
These enzymes can be employed in a large number industrial processes ( production of agrochemicals, cosmetics , biodiesel etc.)
HISTORICAL BACKGROUND: In 1856, Claude Bernard first discovered a lipase in pancreatic juice as an enzyme that hydrolyzed insoluble oil droplets and converted them to soluble products.
In 1901, the presence of lipases has been observed for Bacillus prodigiosus , B.pycocyancus and B.fluorescens which represents today’s best studied lipase producing bacteria now named Serratia marcescens , Pseudomonas aeruginosa and P.fluorescens.
Lipase have traditionally been obtained from animal pancreas and are used as a digestive aid for human consumption either in crude mixture with other hydrolases (pancreatin) or as a purified grade.
Lipolase was the first commercial recombinant lipase industialized from the fungus Thermomycesl anugiwnosus and expressed in Aspergillus oryzae in 1994.
PROPERTIES: pH optima
Temperature optima and thermal inactivation
Activation and inactivation of the enzyme
Substrate specificity
SOURCES: Plant lipases:
These have been isolated from the leaves, oils, latex and seeds of oleaginous plants and cereals.
Yeast Lipases:
These include species Candida antartica, Candida rugosa, Candida utilis and Saccharomyces species. The production of Biodiesel includes lipases from Thermomycesl anuginosus.
Animal Lipases:
These include pancreatic and pregastric lipases.
Porcine and Human pancreas were the first sources of lipases used in food processing.
Bacterial Lipases: The genera Pseudomonas and Burkholderia are the most widely used for the production of bacterial lipases. P.aeruginosa produces a cystiene hydrolase solvent tolerant lipase.
Fungal Lipases:
Filamentous fungi are considered to be the best source for production of lipases. The genera includes Aspergillus, Rhizopus , Penicillium , Mucor, Geotrichum and Yarrowia etc.
PRODUCTIONTECHNOLOGY:
UpstreamProcessing:
Screening
Strain selection
Inoculum preparation
Immobilization
Fermentation :
Solid-State Fermentation
Submerged Fermentation
Downstream Processing:
Filtration
Centrifugation
Chromatography
Aqueous two phase
Raw Materials and Nutrients:Olive oil, Palm oil, Coconut oil
wheat Bran, rice bran
yeast extract, peptone
Urea, NaNO2
Sucrose , glucose , fructose
KH2PO4
MgSO4 .7 H2O
Microbial Sources:
Bacillus sp.
myjunior09.wordpress.com
CONTENTS-
Introduction
• History
• SCP production in India
• Raw materials
• SCP production
• Advantages and Disadvantages
• Applications
• Conclusion
• References
1. Analysis of the Barley Grain
Protease Spectrum
Angela Bell, Peter C. Morris & James H. Bryce
International Centre For Brewing and Distilling,
School of Life Sciences,
Heriot – Watt University, Edinburgh, UK
2. Goals of the Project
• To characterise the protease activity in malted
& germinating barley grains
• To identify individual proteases using
proteomic techniques
• To investigate the significance of specific
proteases in the malting process
3. Overview
• Malting = Germination under controlled
conditions and up to a specific point in
germination process
• Malting stopped at point of grain modification
• Proteases = essential components of
modification process
4. Proteases
• Very important biologically
• Four mechanistic classes:
– Serine
– Cysteine
– Metallo
– Aspartate
• Classes can be differentiated between on the
basis of specific inhibitors
5. Barley Grain Proteases
• Cysteine proteases = the most active protease
class, followed by aspartate, serine then the
metalloproteases
• Cysteine already well characterised:
EP A & EP B purified, analysed & sequenced in
1980’s (Koehler, S., Ho, Tuan – Hua, D (1988), Davy, A., et al
(1998))
• Other classes not so well studied
6. Project So Far. . . .
I. Developed a reproducible & sensitive protease
assay
II. Carried out preliminary protease activity studies
using crude extracts of 4 - day Oxbridge malt,
with & without class specific inhibitors
III. Carried out physiological studies on protease
activated starch degrading enzymes
IV. Worked on method optimisation for protease
purification
11. Physiological Studies
• Three major starch degrading enzymes
putatively influenced by proteolytic activity:
Limit Dextrinase
α – Amylase
β – Amylase
• Assay enzymes in the presence of class
specific protease inhibitors & by germinating
grains in the presence of different
endoprotease inhibitors
12. Limit Dextrinase
• Limit dextrinase = a key starch degrading
enzyme present in germinating barley grains
• Present in an inactive form bound to a
proteinaceous inhibitor molecule
• Proposed to be activated by cysteine protease
mediated breakdown of inhibitory complex
and also reducing conditions
14. α - Amylase
• Is synthesised during germination & is one of
the few enzymes present in the barley grain
during germination that can initiate native
starch hydrolysis
• Due to the presence of α – amylase inhibitors
in the grain, inhibitor degradation is required
for full activity
• Inhibitory complexes thought to be broken
down by protease activity
15. α – Amylase, Four Day Malt:
Inhibitors at Assay Stage
17. Western Blotting
Serine & Aspartate Proteases are +ve regulators of the amount α –
amylase present during grain germination
2 3 4 2 3 4 2 3 4 2 3 4
72 KDa
55 KDa
36 KDa
Day of
Germination
Serine
and
Aspartate
Proteases
Inhibited
Only Aspartate
Proteases
Inhibited
Only Serine
Proteases
Inhibited
Control
19. β - Amylase
• Is synthesised during grain development &
stored in an inactive form bound to a
proteinaceous inhibitor
• Putatively activated during grain germination
by protease activity & / or reducing conditions
• Been suggested that β – amylase degraded by
serine class protease activity
21. β – Amylase During
Germination
0
1
2
3
4
5
6
7
8
Day 1 Day 2 Day 3 Day 4 Day 5
MeanUnitsBeta-Amylase/gFlour
Control
5mM PMSF
Free β - Amylase
22. Conclusions
• limit dextrinase is indeed activated by cysteine class
proteases and reducing conditions, and may require
presence of ion2+
/ metalloproteases for activity
• α – amylase may be activated by aspartate and
serine class proteases
• both aspartate and serine class proteases are
important for the amount of α – amylase present in
grains during germination in a process involving GA3
• β – amylase is degraded by serine class proteases
and requires divalent cations for activity
23. Current & Future Prospects
• Protease purification: many issues!
- Sample stability
- “dirty” samples
- Too many proteins for positive identification
• The future = Investigations into the roles of
aspartate and serine proteases in the
gibberellic acid induced expression of alpha -
amylase
24. • Thank you to:
• Maltsters Association of Great Britain and
Lindisfarne Trust for funding my project
• All in Peter Morris’s Lab, Heriot – Watt
University, Edinburgh
