Protease Production by Fermentation Process, Stages, Microorganisms

Protease enzymes break down proteins in food into amino acids, which are then used by other enzymes to form new proteins essential for the body’s growth and repair. These enzymes are produced in the stomach, pancreas, and small intestine.

Microbial production of protease enzymes is done using solid-state fermentation (SSF) and submerged fermentation (SmF). Proteases are widely used in various industries and biotechnology applications.

They are essential for producing Klenow fragments, synthesizing peptides, digesting unwanted proteins during nucleic acid purification, and in cell culturing.

Microbial Protease Production by Fermentation Process

Definition of proteases

Protease is an enzyme that breaks the peptide bonds present in proteins by hydrolysis reaction.


What are Proteases?

  • Protease is the enzyme that breaks down proteins into smaller polypeptides or single amino acids by the process of proteolysis.
  • Proteases are also called peptidases or proteinases or proteolytic enzymes.
  • Protease enzymes break the peptide bonds of proteins.
  • Protease enzymes are found in all life forms like bacteria, archaea, animals, plants, and some viruses.
  • Proteases can be produced by using microorganisms such as Aspergillus niger, Bacillus subtilis, etc.
  • Protease enzymes are generally involved in various metabolic processes such as photosynthesis, digestion, cell signaling, protein processing, regulation of protein function, apoptosis, and other metabolic processes.

Classification of proteases

  • Based on the catalytic domain Proteases are classified as serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteinases, etc.
  • Based on pH they are divided into acid proteases, neutral proteases, and alkaline proteases.
  • Based on the site of action on protein substrates, they are broadly classified as endo-peptidases or exo-peptidases.

Types of Protease Enzyme and Its Functions

Protease Enzyme NameThe function of the Enzyme
PepsinThis enzyme is Present in the stomach and converts proteins to smaller peptides – proteases and peptones
TrypsinFound in pancreatic juice and breaks proteins and peptones and proteoses into dipeptides
ChymotrypsinFound in pancreatic juice and breaks proteins and peptones and proteoses into dipeptides
ThrombinInvolved in blood coagulation
InsulinasePresent in the kidney and liver. It degrades insulin
HyaluronidasePresent in the acrosome of sperms and helps in the penetration of sperm into the ovum during fertilization
CollagenaseIt digests collagen
ElastasePresent in pancreatic juice and digests elastin
CarboxypeptidaseFound in pancreatic juice and breaks proteins and peptones and proteoses into dipeptides

Substrate Used for Protease Production

Wheat bran, rice bran, sugarcane bagasse, barely bran, corn meal, and corn husk were used as the substrate for the production of protease enzyme by fermentation.

Microorganisms used for Protease Production

  • Different types of microorganisms have different abilities to produce Proteases. Therefore, different microorganisms can be used to produce the proteases that people need. 
  • Today, protease fermentation technology plays a huge role in the food, Pharma, and Medical industry. 
  • To produce proteases a large number of micro-organisms are used like Yeast, bacteria, and fungi. 
  • Proteases are mainly produced by submerged fermentation.
  • Bacillus species are mostly used in the commercial production of proteases eg. Bacillus subtilis
  • The main advantages of using this microorganism are: that it is easier to obtain and easy to handle its ease of handling. It gives maximum yield and can ferment a variety of cheap raw materials like molasses and starch.

Sources for Protease Production

  • Bacterial protease : strains of Bacillus subtilis, Bacillus licheniformis etc.
  • Fungal Proteases: Commercial production of fungal protease- Aspergillus flavus, Aspergillus wentii, Aspergillus oryzae, Mucor miehei, Mucor Delmar, and Amylomyces rouxii, etc.
  • Animal Protease: The most common animal proteases are pancreatic pepsin, trypsin, chymotrypsin, and rennins. Pepsin- present in the stomachs of vertebrates, Trypsin from the intestinal digestive tract, Chymotrypsin from the animal pancreatic extract.
  • Plant proteases: Papain, bromelain, and ficin represent some of the well-known plant proteases. Papain from papaya fruits, Aspartic protease from potato leaves, Bromelain from stem and juice of pineapples, and Thiol Protease from pineapple Crown Leaf.

Techniques of Protease Production

1. Solid state fermentation: Solid-state fermentation (SSF) is an industrial process, in which metabolites are mostly generated by microorganisms grown on solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes ( bread, maturing of cheese).

2. Submerged fermentation: Submerged fermentation is an industrial process in which enzymes and other reactive compounds are submerged in a liquid such as alcohol, or a nutrient broth. The process is used for a variety of purposes, including in the industrial manufacturing of protease enzymes.

Stages of Protease Production

Industrial fermentation of proteases requires the three most important stages to complete product fermentation. These are upstream processes, actual fermentation and last one is downstream processes.
1. Upstream  process of fermentation:
  • The upstream process includes the initial steps of a fermentation process.
  • The Up-Stream process of protease fermentation comprises initial tasks to screening and identification of suitable microorganisms, and their improvement, Substrate selection, and Culture media preparation to enhance productivity and yield. 
  • It also includes the maintenance of strain purity, preparation of inoculum, and Sterilization of media and bioreactor to improve the economic efficiency of the process.
2. Actual Fermentation Process:
  • Fermentation is a process in which complex substances are converted into simple substances with the help of microorganisms.
  • In this process, all the materials are added together in a bioreactor to produce the desired product.
  • Maintaining optimal conditions required for citric acid production lilk nutrients, are supplied for the growth and multiplication of microorganisms inside a bioreactor, Temp, pH, Oxygen concentration, etc.
3. Down-stream process:
  • The downstream process of fermentation involves the recovery of products from the bioreactor by different processes like extraction, separation, purification, and packaging of the product.

Factors Affecting Industrial Protease Production

1. Temperature : 
  • The effect of temperature on protease enzyme production is numerous. 
  • The optimum temperature for the protease activity was observed at 40°C with substantial activity between 30 and 50°C. At 50 and 60°C, the activity decreased to 97% and 85%, respectively.
  • Temperature can affect the growth of microorganisms’ activity. In the optimum temperature range, as the temperature increases the growth and metabolism of the microorganisms accelerate, and the rate of the fermentation reaction increases. 
  • When the temperature exceeds the optimal temperature range temperature rises the microorganism’s activity is quickly inactivated and the fermentation rate is reduced. 
  • Therefore, to ensure a normal fermentation process, it is necessary to maintain the optimum temperature.
2. pH : 
  • pH can affect the activity of microorganisms, enzymes, and fermentation rate. 
  • Based on the type of substrate and media and microorganisms the pH is vary. 
  • The optimum activity of crude protease extract was found at pH 7.0, Optimum pH of intestinal proteases was between 9 and 11.  
  • In addition, pH will also affect the decomposition of nutrients in the medium. Therefore, the pH of the fermentation broth should be controlled.
3. Dissolved oxygen Concentration : 
  • The supply of oxygen is a critical factor for aerobic fermentation and Aspergillus niger is an aerobic microorganism and therefore requires oxygen. 
  • Aeration and agitation meet the oxygen demand of the fermentation process of citric acid.
  • Agitation is important for heat transfer, adequate mixing, and mass transfer.
  • Aeration and agitation both maintain the homogeneous environment of culture by continuous mixing.
  • Therefore, a large amount of oxygen must be continuously added to the fermentation broth, and stirring can increase the solubility of oxygen in the fermentation broth. The concentration of dissolved oxygen increased with the increase in the speed of agitation.
4. Concentration of nutrients : 
  • The concentration of various nutrients in the fermentation broth, especially the ratio of carbon to nitrogen, inorganic salts, and vitamins, will directly affect the growth of bacteria and the accumulation of metabolites.
  • Such as Carbon sources such as Starch, glucose, sucrose, fructose, maltose, and mannose. nitrogen sources like ammonium or nitrate ions. And other nutrients like phosphate, low amounts of potassium, magnesium, sulfate, and trace metals such as iron, manganese, zinc, and copper.
  • For example, in the fermentation of glutamate, the change in NH+4 concentration will affect the metabolic pathway. 
  • Therefore, a sufficient amount of nutrients are added for fermentation during the fermentation process.

FAQs on Protease Production by Fermentation

1. How are proteases produced?
Answer: Industrial Proteases are mainly produced by microorganisms using submerged and solid-state fermentation techniques. 
2. Does yeast produce protease?
Answer: Extracellular proteinases secreted by yeasts have been isolated and examined by several researchers.
3. Where is a protease produced? 
Answer: Protease is produced in the stomach, pancreas, and small intestine.
4. Can bacteria produce enzyme protease?
Answer: Yes, A wide variety of bacterial species produces protease enzymes such as Bacteroides bivius, Bacteroides fragilis, Staphylococcus aureus, Staphylococcus epidermidis, etc. 
5. Which microorganisms are used to produce protease?
Answer: Microorganisms including Bacteroides bivius, Bacteroides fragilis, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa, produce various proteases.
6. What are the types of proteases?
Answer: Based on the mechanism of catalysis, proteases are classified into six distinct classes, aspartic, glutamic, and metalloproteases, cysteine, serine, and threonine proteases

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