This document discusses proteases and their industrial applications. It begins by introducing proteases as one of the most widely studied marine enzymes, which account for over 60% of the global enzyme market. It is used in many industries like food processing, detergents, leather, and pharmaceuticals. The document then describes the three main types of proteases - alkaline, neutral, and acid proteases. It provides examples of marine microbial proteases that have properties making them suitable for industrial applications. The document concludes by summarizing several studies that have optimized production and properties of novel alkaline proteases with potential uses in detergents and pharmaceuticals.

1. PROTEASES
P 4 U 2
Vedanti S. Gharat
T.Y. B.Sc. Biotechnology
Roll No. : 20

2. INTRODUCTION
• Proteolytic enzymes account for
above 60% of the global enzyme
market, and proteases are one of
the most widely studied marine
enzymes.
• They are vastly used in the food
processing, detergent, leather,
and pharmaceutical industries,
and in biotechnological
research. They have potential
applications in bioremediation
and waste management.
• The increased demand for
industrial proteases over its
supply from plant and animal
sources led to an increased
interest in microbial proteases.
70% of industrial proteases
come from microbial sources.

4. TYPES OF PROTEASES
1. Alkaline proteases
2. Neutral proteases
3. Acid proteases
• Alkaline and Neutral proteases are obtained mainly from
bacterial sources, mostly from different species of Bacillus.
• Fungi are a well-known source of Acid proteases.
• Marine microbial proteases gained attention for their
extremophilic properties and stability in the presence of a
broad range of chemicals.
• They possess almost all the characteristics desired for
various biotechnological applications of proteases.

5. • A novel alkaline serine protease was reported from the marine
fungus Engyodontium album BTMFS10 strain isolated from
marine sediment from the west coast of India.
• The extracellular enzyme was produced by solid-state
fermentation and the process parameters of protease production
were optimized.
• The enzyme was active over a broad range of pH values (6-12)
and temperature ranges (15-65°C) and retained its activity in the
presence of hydrocarbons, natural oils, surfactants, and organic
solvents.
• The optimum activity of the purified enzyme at a high
temperature (60°C) and pH value (pH 11) suggest its potential
application in detergent industry.

6. • Sana et al. reported another alkaline serine protease from a marine-
proteobacterium isolated from the intertidal zone of the Bay of
Bengal.
• The enzyme was tolerant to salt, solvent, detergent, and bleaching
agents, and showed potential for the detergent industry and dry
washing.
• A protease encoding gene was isolated from Engyodontium album,
which encodes a protein with 96% sequence similarity to proteinase
R of Tritirarchium album.
• A homology comparison of the amino acid sequence revealed that
this protease belongs to the subtilase family of serine protease.
• The enzyme structure was reported to be stabilized by two disulfide
bonds and more than two Ca²+ binding sites, which are assumed to
contribute to the thermostability of the enzyme.

7. • Surface-displaying recombinant alkaline proteases were
produced on a Yarrowia lipolytica cell surface by
overexpressing a marine-derived alkaline protease gene.
• This surface-displaying protease can hydrolyze various
proteins for the production of bioactive peptides.
• The peptides have potential pharmaceutical applications due to
their angiotensin-converting enzyme (ACE) inhibitory
activity and antioxidant activity.
• Peptides with high ACE inhibitory activity have well known
potential application in the treatment of hypertension and other
bioactive peptides can be used as clinical nutrient
supplements.

8. • The production of a thermostable alkaline protease by a
marine Bacillus was optimized and scaled up in a stirred
tank bioreactor using cheap and readily available
substrates.
• Enzyme production increased with prolonged
fermentation time and faster agitation rate.
• Protease production by the marine bacterium
Teredinobacter turnirae was optimized for solid-state
fermentation as well by immobilization on solid matrixes.
• The material and particle size of the immobilizing
medium, and the method and conditions for cell
immobilization were also optimized for repeated batch
fermentation of the alkaline protease by T. turnirae.

9. • Ceramic support, the different sizes of broken pumice stone,
and silicone foam enhanced enzyme production by more than
200%, when compared to free cells.
• Protease production increased with an increased number of
fermentation cycles and was maximized at the fourth cycle
when the enzyme activity reached about 3:5 times more than
that of the first cycle.

10. Reference
• Springer Handbook of Marine Biotechnology by Kim, Se-
Kwon.
• Images from Google.