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Isolation and Characterization of Protease from Marine Algae

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Jeyanthi Rebecca at Bharath University
Jeyanthi Rebecca
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Lipases are one of the widely used biocatalyst for various purposes and so they have wide range of applications. These water soluble enzymes have unique ability of catalyzing the hydrolysis of ester bonds in triacylglycerols. Lipase is the primary digestant used to break fats into fatty acids and glycerol. Lipase is produced by different methods and via variety of sources. Isolation of lipase is a challenging task as it should be cost effective and efficient one at industrial scale. Lipase has been isolated from marine algae which is easily available in shallow water and from culturing of the same. People are not using seaweeds as a food in India so it gives us a better chance and challenge to utilize it for different purposes. In this research work we isolated lipase from different types of seaweeds from nearby area, purified it and estimated the molecular weight of lipase. The obtained lipase was tested against different physical and chemical parameters such as effect of pH on activity and stability, effect of temperature on activity and effect of different substrates on the activity of lipase showing its optimum range and specificity for different substrates.
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Int. J. Pharm. Sci. Rev. Res., 27(1), July – August 2014; Article No. 34, Pages: 188-190 ISSN 0976 – 044X
International Journal of Pharmaceutical Sciences Review and Research
Available online at
www.globalresearchonline.net
© Copyright protected. Unauthorised republication, reproduction, distribution, diss emination and copying of this document in whole or in part is strictly p rohibited.
© Copyright protected. Unauthorised republication, reproduction, distribution,
188
Swapnil Suresh Patil, L. Jeyanthi Rebecca*
Department of Industrial Biotechnology, Bharath University, 173, Agharam Road, Selaiyur, Chennai, Tamilnadu, India.
*Corresponding author’s E-mail: hodbiobharath@gmail.com
Accepted on: 29-04-2014; Finalized on: 30-06-2014.
ABSTRACT
A protease is a member of a very large group of enzymes that have various functions in the body. Proteases break the long chainlike
molecules of proteins into shorter fragments. Proteases are found in plants, animals, including microorganisms such as fungi,
bacteria and yeast. Protease is considered to be an important industrial enzyme accounting for about 60% worldwide sale of
enzymes. The influence of parameters such as temperature, pH were evaluated.
Keywords: Algae, Electrophoresis, Protein content, Protease, purification.
INTRODUCTION
he ocean is considered to be the repository of many
unexploited natural resources. Protease hydrolyzes
the peptide bonds and is also termed as
proteinase.1 Protease constitutes one of the most
important groups of industrial group having application in
different sectors of industries. Protease is widely being
used in waste management, silver recovery from X-ray
films, leather industry, and detergent industry.2-4
Proteases have found applications in molecular biology
research as well.5 A protease from Bacillus
amyloliquefaciens may be used to promote flavor
production in cheddar cheese.6 Proteases are also being
used in meat processing, dairy, digestive aid7 Protease
has found an irreplaceable role in silk industry.8,9
Reports are available on protease production by fungal
species belonging to Aspergillus genera.10 There are
reports on the isolation of protease from plant leaves and
marine waste like fish scales, crab and prawn shells.11-14
MATERIALS AND METHODS
Estimation of protein content by Bradford Method
The protein concentration was measured by the Bradford
method, using BSA as a standard. Dilute the Bradford
reagent and add the sample to the diluted reagent. The
colour was changed to dark blue. The absorbance was
taken at 590 nm.15
Protein precipitation using Ammonium sulphate
Ammonium sulphate was used to precipitate the protein.
Appropriate amount of ammonium sulphate is added to
the supernatant and centrifuged for 10 mins at 10000
rpm. The pellets were resuspended in suitable buffer.
Partial purification by Sephadex column
Sephadex was used to pack the column. The solution was
fed to the column for further purification.
Protease activity
0.5 ml of partially purified enzyme was mixed with 0.5 ml
casein. The reaction mixture was incubated for 1 hr at
37°C and stopped by addition of 1 ml of 10% TCA. The
mixture was centrifuged and supernatant was collected.
0.5 ml of supernatant was mixed with Na2CO3 and NaOH
and Folin Phenol. This mixture was shaken well and OD
was taken at 650nm. One protease unit is defined as the
amount of enzyme that releases 0.5 µg/ml/min tyrosine.
The protease activity was measured
spectrophotometrically.
Effect of pH and Temperature
The optimum pH of the enzyme was determined using
buffer at varying pH ranges of 3-12. The effect of
temperature on the protease activity was studied by
incubating the reaction mixture at different temperature
ranging from 20-90°C.
Sodium Dodecyl Sulphate-Polyacrylamide gel
electrophoresis (SDS-PAGE)
SDS-Polyacrylamide gel electrophoresis was performed
on slab gel with separating and stacking gels by the
method of (Laemmli, 1970).16
RESULTS AND DISCUSSION
The present study was carried out to isolate and
characterize protease from algae. The protein content of
all the samples is shown in Table 1.
Table 3 and table 4 clearly show that parameters like
temperature and pH affect the activity of enzyme.
Maximum species showed highest activity at 40°C. The
optimum pH was checked for all the samples at varied pH
range. The optimum pH range was observed between 7-
7.5 depending on species, although the maximum activity
was seen at pH 7.2.
It can be observed from the above result that the isolated
enzyme from different species had molecular weight
Isolation and Characterization of Protease from Marine Algae
T
Research
Article
Int. J. Pharm. Sci. Rev. Res., 27(1), July – August 2014; Article No. 34, Pages: 188-190 ISSN 0976 – 044X
International Journal of Pharmaceutical Sciences Review and Research
Available online at
www.globalresearchonline.net
© Copyright protected. Unauthorised republication, reproduction, distribution, diss emination and copying of this document in whole or in part is strictly p rohibited.
© Copyright protected. Unauthorised republication, reproduction, distribution,
189
within the range of 30-45 kDa. The bands were compared
with the standard bacterial protease run alongside.
Table 1: Protein estimation of crude and purified samples
Species Name Protein content of
crude extract
(µg/ml)
Protein content
(purified)
(µg/ml)
Ulva lactuca (Puducherry) 92 82
Ulva fasciata (Puducherry) 97 82
Enteromorpha compressa
(Puducherry) 86 77
Chaetomorpha antenna
(Puducherry) 105 90
Ulva lactuca
(Mahabalipuram) 92 77
Enteromorpha
flexuosa(Mahabalipuram) 110 90
Table 1 shows the protein content of 6 species of
seaweed. The highest protein content was seen in
Enteromorpha flexuosa (Mahabalipuram) and
Chaetomorpha antenna (Puducherry) while lowest was
found in Ulva lactuca (Mahabalipuram) and
Enteromorpha compressa (Puducherry).
Table 2: Activity of protease using Casein as substrate
Species Name Place
Protease activity
(units/mg)
Ulva lactuca Puducherry 7.33
Ulva fasciata Puducherry 8.0
Enteromorpha
compressa Puducherry 6.74
Chaetomorpha
antenna Puducherry 9.4
Ulva lactuca Mahabalipuram 6.55
Enteromorpha
flexuosa Mahabalipuram 9.6
The above Table shows Enteromorpha flexuosa showing
the highest activity while Ulva lactuca (Mahabalipuram)
showed the lowest.
Table 3: Effect of Temperature on Protease activity
Species Temperature Range (°C)
20 30 40 50 60 70
Ulva lactuca 6.33 6.71 7.35 6.4 5.20 4.42
Ulva fasciata 6.92 7.31 8.0 7.37 5.32 3.9
Enteromorpha
compressa 5.22 6.35 6.77 6.20 4.39 3.55
Chaetomorpha
antenna 6.78 8.42 9.49 8.11 6.22 5.14
Ulva lactuca 4.52 5.91 6.61 5.33 4.59 4.38
Enteromorpha
flexuosa 6.55 8.61 9.38 7.52 6.17 5.21
Table 4: Effect of pH on Protease activity
Species pH range
6.5 7.0 7.5 8
Ulva lactuca 6.21 7.25 7.41 6.79
Ulva fasciata 6.42 8.0 8.22 7.66
Enteromorpha compressa 5.85 6.71 6.77 5.73
Chaetomorpha antenna 8.22 9.12 9.22 8.76
Ulva lactuca 5.73 6.43 6.47 5.95
Enteromorpha flexuosa 8.46 9.48 9.59 8.66
Figure 1: Estimation of molecular weight of algal protease
using SDS-PAGE
Note: Lane 1 - Ulva lactuca (Puducherry); Lane 2 - Ulva fasciata
(Puducherry); Lane 3 – Ulva lactuca (Mahabalipuram); Lane 4 –
Enteromorpha compressa (Puducherry); Lane 5 – Chaetomorpha
antenna (Puducherry); Lane 6 – Enteromorpha flexuosa
(Mahabalipuram); Lane 7 – Standard bacterial Protease
CONCLUSION
The major aim of the study carried out was to investigate,
characterize and purify the protease enzyme from various
marine algae. The enzyme extracted from different
marine algae showed positive results for protease assay
confirming the presence of protease. The activity varied
with different species and temperature and pH.
From this study, it is quite evident that marine algae are a
valuable source of Protease. Moreover, they can also be
screened for other industrially important enzymes.
REFERENCES
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3. Fujiwara N, Tsumiya T, Katada T, Hosobuchi T, Yamamoto
K, Continuous Recovery Of Silver From Used X-Ray Films
Using A Proteolytic Enzyme, Process Biochem, 24, 1989,
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4. Babu NKS, Lakshmi KD, Afr.J.Biotechnol., 4(7), 2005, 724-
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Int. J. Pharm. Sci. Rev. Res., 27(1), July – August 2014; Article No. 34, Pages: 188-190 ISSN 0976 – 044X
International Journal of Pharmaceutical Sciences Review and Research
Available online at
www.globalresearchonline.net
© Copyright protected. Unauthorised republication, reproduction, distribution, diss emination and copying of this document in whole or in part is strictly p rohibited.
© Copyright protected. Unauthorised republication, reproduction, distribution,
190
5. János András Mótyán, Ferenc Tóth, József Tőzsér, Research
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Source of Support: Nil, Conflict of Interest: None.
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Full-text available
  • Oct 2012
Industrial enzymes are produced by various organisms using a wide variety of substrates. The present study was carried out to investigate the production of protease by Aspergillus niger using fish scale as the growth media. Fish scale, the chief waste material of fish processing industries, was enzymatically hydrolysed by protease produced by A. niger. Two mutated strains of A. niger, namely, AB30 and AB60 were also used apart from the wild type A. niger strain. Trichloroacetic acid buffer was used to maintain the optimum moisture content of the substrate. No separate nutrient sources apart from the fish scales were used. This proves the cost effectiveness and efficiency of this process. The enzyme activity was analyzed from the 5 th day and it was further analyzed for three consecutive days and the results were plotted and analyzed.
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Protease Enzyme-Potential Industrial Scope
Article
  • Nov 2011
Enzymes are vitally important for the existence of life. Civilizations have used enzymes for thousands of years without understanding what they were or how they work. Over the past several generations, science has unlocked the mystery of enzymes and has applied knowledge to make better use of it in an ever growing number of applications. Enzymes play crucial role in producing the food we eat, the clothes we wear and even in producing fuel for our automobiles. They are important in combating environmental pollution. Leather processing is one of the important industries closely related to everyday life. Proteases execute a large variety of functions and have important biotechnological applications. They represent one of the largest groups of industrial enzymes and find application in detergents industry, leather industry, food industry, pharmaceutical industry and bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al. 2002). Probably the largest application of proteases is in laundry detergents, where they help removing protein based stains from clothes (Banerjee et al. 1999). In textile industry, proteases may also be used to remove the stiff and dull gum layer from the raw silk fibre to achieve improved luster and softness. They can modify the surface of wool and silk fibres to provide new and unique finish. Proteases have been used in the hide-dehairing process, where dehairing is carried out at pH values between 8 and 10.
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