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Isolation of protease producing bacterial strain from Jazan

province and study of effect of the temperature on the production

of enzyme By

Ismaeil Hassan Ahmed Melhan

Univ.No.201011368

B.Sc. student, Biology Department

Faculty of Science, Jazan University

Abstract

Samples were collected from different places including marine mangrove

rhizophers and garden soil from Jazan shores. Skim milk nutrient agar media

was used for qualitative screening for protease using streaking method.

Colonies forming transparent zones, because of partial hydrolysis of milk

casein, were selected. After being incubated for 24 hrs, a plate containing

milk and agar showed the growth of several colonies. The zone formation

around the bacterial colony indicated the protease positive strain which may

be due to hydrolysis of casein. It was found that one strain showed the

highest number of enzyme producers followed by the clear zone. It was

observed when incubated over night at 37°C more than incubation on 30 and

45°C respectively. The potent protease producer was found in G+ve

Bacterium isolate.

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1.Introduction

1.1. Mangroves

Mangroves are various kinds of trees up to medium height and shrubs that

grow in saline coastal sediment habitats in the tropics and subtropics mainly

between latitudes 25° N and 25° S. The remaining mangrove forest areas of

the world in 2000 was 53,190 square miles (137, 760 km²) spanning to 118

countries and territories. The mangrove biome, or mangal, is a distinct saline

woodland habitat characterized by a depositional coastal environments,

where fine sediments (often with high organic content) collect in areas

protected from high-energy wave action. Mangroves dominate three quarters

of tropical coastlines. The saline conditions tolerated by various mangrove

species range from brackish water, through pure seawater, to water

concentrated by evaporation to over twice the salinity of ocean seawater [1].

1.2. Thermophilic Bacteria

A thermophile is an organism a type of extremophile that thrives at

relatively high temperatures, between 45 and 122 °C (113 and 252 °F).

Thermophilic eubacteria are suggested to have been among the earliest

bacteria. Thermophiles are found in various geothermally heated regions of

the earth, such as hot springs and deep sea hydrothermal vents, as well as

decaying plant matter, such as compost. As a prerequisite for their survival,

thermophiles contain enzymes that can function at high temperatures. Some

of these enzymes are used in molecular biology (for example, heat-stable

DNA a polymerases for PCR), and in washing agents (for example

proteases, amylases and lipases). Thermophiles are classified into obligate

and facultative thermophiles: Obligate thermophiles (also called extreme

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thermophiles) require such high temperatures for growth, whereas

facultative thermophiles (also called moderate thermophiles) can thrive at

high temperatures, but also at lower temperatures (below 50°C) [2].

1.3. Bacterial proteases

Bacterial extracellular protease has been studied more extensively in Gram

positive bacteria. The well characterized enzymes of this group are the

subtilisins of B. subtilis, the pronases of Streptomyces grise, collagenases

and clostripain from Clostridium histolyticum, and streptococcal proteinases

of group (A) Streptococci. Table (1) summarizes various proteolytic

enzymes produced by bacteria [3].

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Table (1): Representative examples of proteolytic enzymes produced by

bacteria

Microorganism Type Substrates

Gram-positive Bacteria:

Arthrobacter sp. Serine,alkaline

Wide range (e.g. casein,

gelatin, haemoglobin)

B.cereus Acid, chymosin- like Casein

B.cereus Neutral, EDTA senstive

Casein

B. licheniformis

Serine, alkaline and

neutral, EDTA senstive

Heamoglobin, casein

B. megaterium Neutral, EDTA

senstive Casein

B. sphaericus Serine, alkaline, EDTA sensitive

Casein

B. subtilis

Serine, alkaline,

(Subtilisin). Neutral, EDTA-sensitive

Wide range (e.g. casein, elastin)

Streptococci Thiol Casein, fibrin M protein,

gelatin

Gram-negative Bacteria:

Aeromonas proteolytica

Neutral, EDTA-

sensitive

Casein, gelatin,

heamoglobin

Escherichia freundii Alkaline, EDTA-

sensitive Casein

Pseudomonas fragi Neutral, EDTA-sensitive

Casein

Serratia sp. Alkaline, EDTA-sensitive

Casein, insulin heamoglobin

Adopted from Elgayar (2001) [3] .

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1.4. Uses of Proteases

Most of the practical applications of proteases have been developed by

empirical methods for selecting the most suitable protease or combination of

proteases and the most appropriate conditions of use [4]. The following are

some of the applications that use proteases:

1. Meat tenderization

Meat tenderization is done by injecting proteolytic enzymes into the vascular

system either before or after slaughter. Proteases are also used to recover

protein from parts of animals and fish that would otherwise go to waste after

butchering.

2. Dairy industry

Lactic acid bacteria constitute a very important group in the dairy industry

for the production of fermented milk products such as cheese. These bacteria

are solely depending on their proteolytic system for the degradation of

casein, which also meets their demand for a supply of peptides.

3. Pharmaceutical industry

Proteases from Aspergillus oryzae have been used as a digestive aid to

correct certain lytic enzyme deficiency syndromes. Table (2) summarizes

some applications of the proteases in the medical field.

4. Tanning of leather

This process is normally carried out by treating animal hides with a saturated

solution of lime and sodium sulphide. Besides being expensive and

particularly unpleasant, a strongly polluting effluent is produced.

Alternatively, enzymes can be applied. Enzyme assisted dehairing method is

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possible if proteolytic enzymes are stable and active under alkaline

conditions (pH12).

Table (2): Some applications of proteases in the medical field

Enzyme Source Effect

Bromelain Pineapple Digestive aid and reduction of oedema

Chymopapain Papaya Treatment of herniated discs

Chymotrypsin Pancreas Treatment of inflammatory condition

Ficin Fig Digestive aid

Kallikrein Pancreas Reduce blood pressure

Papain Papaya Digestive aid, reduction of oedema and treatment of

oedema

Pepsin Mammalian stomach Digestive aid

Plasma Human blood Dissolve blood clots

Protease Bacterial Digestive aid

Streptokinase Human urine Dissolve blood clots

Trypsin Pancreas Digestive aid , treat athletic injuries

Urokinase Human urine Dissolve blood clots

* Adopted from Elgayar (2001) [3].

5. Degradation of proteinous waste into useful compounds by proteases

Wastes rich in protein such as feathers, carrion, and hide scrap are suitable

sources. Through technological processes employing proteolytic enzymes

these wastes can be converted into protein concentrates for animal feed.

6. Detergents

One of the major industrial applications of enzymes is in the detergent

industry. More than 95% of the enzymes sold for laundry detergents are

alkaline serine proteases from Bacillus sp., especially the Bacillus subtilis

group. The enzymes were stable in high salt concentrations, heavy metals,

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chelating agents, pH’s up to 11.9 and high temperatures. These

characteristics suggested that the protease should have utility for several

cleansing applications.

1.5 Proteases: their importance and sources

Proteases represent one of the three largest groups of industrial enzymes and

account for about 60% of the total worldwide sale of enzymes (Fig1).

Proteolytic enzymes catalyze the cleavage of peptide bonds in polypeptides

and proteins and resolve racemic mixtures of amino acids [5].

Proteases are physiologically necessary for living organisms, they are found

in a wide diversity sources such as plants, animals, and microorganisms.

Papain , bromelain, and keratinases represent the known proteases of plant

origin. The use of plants as a source of proteases is governed by several

factors such as the availability of cultivated land and the suitability of

climatic conditions for growth. The most familiar proteases of animal origin

are trypsin, chymotrypsin, pepsin, and rennins. Their production depends on

the availability of livestock for slaughter which is governed by political and

agricultural policy. The inability of the plant and animal proteases to meet

current world demands has led to an increased interest in microbial

proteases. Microorganisms represent an excellent source of enzymes owing

to their broad biochemical diversity and their susceptibility to genetic

manipulation. Microbial proteases account for approximately 40% of the

total worldwide enzyme sales. Proteases from microbial sources are

preferred since they possess almost all the characteristics desired for their

biotechnological applications [6].

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Figure (1):

Distribution of enzymes sales. The contribution of different enzymes

to the total enzyme sale is indicated. The shaded portion indicates the

total sale of proteases. Adopted from Elgayar, 2001[3]

Other

Carbohydrases

Analytical and Pharmaceutical

Enzymes

Alkaline protease

Trypsin Rennine

Lipases

Other

proteases

Amylases

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2. Materials and methods

2.1. Media

Nutrient agar medium containing 5 g peptone and 3 g beef extract 5 g NaCl

and 15 g agar agar per liter was prepared [7]. The pH was adjusted to pH 7.0

by NaOH. Skim milk nutrient agar; is nutrient agar supplemented with 5%

skim milk. Skim milk was sterilized separately and mixed just before

solidification. Physiological saline solution contained 8.5 g NaCl per liter

[8]. All media, physiological saline solution and tools were sterilized at 115

°C using autoclave.

2.2. Isolation of bacteria

Samples were collected from different marine places including marine

mangrove rhizophers and garden soil from Jazan shores. One gram of

samples was added to a glass tube containing 25 ml sterilized distilled water.

Serial dilution in physiological saline solution has been done for mixed

samples and each dilution was used to inoculate the nutrient agar medium by

plating method as described by Aftab et al. (2006) [9]. Agar plate medium

containing 25ml solidified medium was inoculated with 100 microliter from

each dilution. These plates were incubated at 37°C for 24 h. The plates that

showed considerable single colonies were selected for this purpose.

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Figure (2):

Shows the area of isolation; at Jazan shores mixed from mangroves

Rhizophores and soil.

2.3. Qualitative screening of protease enzyme strain

Skim milk nutrient agar media was used for qualitative screening for

protease using streaking method. It used to detect protease-producing

isolates [10]. Colonies forming transparent zones, because of partial

hydrolysis of milk casein, were selected. Purified cultures of selected

isolates were streaked on Nutrient agar slants and stored at 4°C.

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2.4. Demonstration of Gram staining

Preparation of a bacterial smear.

10 μl of sterile water in the center of a clean glass slide was placed. A pure

protease-producing bacterium was mixed with water drop on slide. The

water-bacteria mixture was spreaded over an area of about 1 inch square

then allowed to air dry. The slide was hold with forceps and heated on the

hot plate for several minutes [11].

Staining

The smear was covered with a few drops of crystal violet and left for 1

minute. The slide was washed carefully over the top with distilled water

until no large amounts of color wash off. The smear was covered with

Gram’s iodine and left for 1 minute. The smear was decolorized with 95%

ethyl alcohol (Decolorizer) then the slide was washed carefully over the top

with distilled water from a wash bottle until no large amounts of color wash

off. Immediately the slide was rinsed with distilled water. The smear was

covered with safranin and left for 1 minute followed with gently rinsed with

distilled water then blot dried with a paper towel. The specimen was

observed under the microscope to differentiate is it Gram positive or

negative bacterium Fig (3) [11].

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2.5. Monitoring the production of protease at different

temperature

The activity of the extracellular protease Bacterial cells was activated by

growing them overnight on milk agar plate at 37 °C. Ten ml of nutrient

broth medium was inoculated with several recently growing colonies (fresh

colonies) after which cells were allowed to grow for 24 hours at 37 °C with

shaking at 150 rpm. Three flasks, each one containing 100 ml of soyabeab

broth medium were inoculated with 2ml of the above culture. The new

Figure (3):

The steps of gram staining for Bacteria isolates

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culture was allowed to grow at 30°C, 37 °C and 45 °C with shaking for 2

days. At the indicated time, 100µl of the growing culture was taken and

centrifuged in a microcentrifuge at 2500 rpm for 2 minutes. The

supernatants were then used as crude enzyme to determine the activity of

protease.

3. Results and discussion

Isolation and screening of bacterial strains for protease

During the recent years, efforts have been directed to explore the means to

reduce the protease production cost through improving the yield, and the use

of either cost free or low cost feed stocks [12]. Isolated bacterial strains were

screened for protease producing ability on skim milk agar. After being

incubated for 24 hrs, a plate containing milk and agar showed the growth of

several colonies. The zone formation around the bacterial colony indicated

the protease positive strain which may be due to hydrolysis of casein. Hence

the strains were identified as a protease producer and it was taken for further

experimental studies. Figure 4 shows the total bacterial strains isolated from

3 different marine places from Jazan shores; soil, Sea water and rhizophores

of mangroves trees incubated overnight on 37°C as described in the

materials and methods. Screened qualitatively using skim milk agar plates. It

was found that one strain showed the highest number of enzyme producers

followed by the clear zone. It was observed when incubated over night at

37°C more than incubation on 30 and 45°C respectively but in the same time

bacteria was tolerant under all temperatures (Figure 5 and 6). The potent

alkaline protease producer (Figure 5B) was found in G+ve Bacterium isolate

(Figure 7).

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Fig 4:

(A)Represents isolation the mixed sample on milk agar with 10-1

dilution. (B) Represents isolation the mixed sample on Nutrient agar

with 10-5 dilution.

A B

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Fig (5):

Represents Qualitative screening for protease by G+ve bacterium

isolate at 30 °C, (B) Represents Qualitative screening at 45°C and (C)

Represents Qualitative screening at 37 °C using skim milk nutrient

agar medium respectively.

A

B

C

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Fig (6):

Represents isolated bacteria growing on skim milk nutrient agar

medium and showing the hydrolyzing the milk, where (1 and 4)

represents growing at 30 °C, (3) Represents growing at 45°C and (2)

Represents growing at 37 °C.

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Fig. (7):

Shows that; the protease bacterium isolate from Jazan; halophilic and

thermophilic mixed from marine, mangrove rhizophores and garden

soil is gram +ve bacilli.

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4. References

1-Giri, C. (2011): Status and distribution of mangrove forests of

the world using earth observation satellite data. Glob. Ecol. Biogeogr.

20, 154-159.

2-Takai T. (2008): Cell proliferation at 122°C and isotopically heavy

CH4 production by a hyperthermophilic methanogen under high-

pressure cultivation. PNAS.105 (31):10949–51.

3-El-Gayer, KH. (2001): Bioconversion of chicken feather waste into

useful products through a recombinant bacterial strain. M. Sc. Thesis,

Department of Bioscience and Technology, Institute of Graduate

Studies and Research, University of Alexandria.

4-Underkofle, L.A. (1976): Microbial enzymes. Industrial

Microbiology, Miller, B.M., and W. Litsky P 135-136, McGaw – Hill

Book Company. Newyork .

5-Anwar, A., and M. Saleemuddin (1998): Alkaline proteases: A

review. Bioresource Technology. 64: 175 – 183.

6-Rao, M.B., A.M. Tanksale, M.S. Ghatge, and V.V. Desh pande

(1998): Molecular and biotechnological aspects of microbial

proteases. Microbiology and Molecular Biology Reviews. 62 (3): 597

– 635.

7-Ganesh, A., S., Swarnalatha, S., Gayathri, N., Nagesh, and G.,

Sekaran (2008): Characterization of an alkaline active – thiol forming

extracellular serine keratinase by the newly isolated Bacillus pumilus.

Journal of Applied Microbiology 104:411–419

8-Pelczar, M.J., and E.C. Chan (1977): Laboratory Exercises in

Microbiology, 4th edition, McGraw Hill, Inc.

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9-Aftab, S.; Ahmed, S., Saeed, S. and Razoo, S.A. (2006):

Screening, isolation and characterization of alkaline protease

producing bacteria. Pak. J. Biol. Sci. 9:2122-2126.

10-Hanaa A. El-Shafei1, Mohamed S. Abdel-Aziz1, Mohamed F.

Ghaly and Ahmed A. H. Abdalla(2010): Optimizing some factors

affecting alkaline protease production by a marine bacterium

Streptomyces albidoflavus. Proceeding of fifth scientific

environmental conference, Zagazic Uni., 125 - 142

11-Maryland University. (2000): The Gram Stain. Pathogenic

microbiology.

http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/Gr

amStain.htm

12-Kuberan, T., S. Sangaralingam, and V.Thirumalaiarasu

(2010): Isolation and optimization of Protease producing Bacteria

from Halophilic soil. J. B iosci. Res., 2010. Vol. 1(3):163-174

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المستخلص العربى

من ع عيناتيجمتم ت .بعزل سالالت بكتيرية لها القدرة على انتاج االنزيم انزيم البروتييز تم إنتاج

البحري المانغروف عينات من الريزوفور لشجر 3 تم خلط .ساحل مدينه جازان من أماكن مختلفة

الحليبب سط الغذائى المدعمالو واستخدمت .جازان و من مياه الشاطىء شواطئ منتربة الحديقة و

زراعة البكتيريا بطريقة التخطيط على اطباق باستخدام طريقة زيلبروتيافحص ل جاراآل و المقشود

تم . الحليبكازين ل التحلل، وذلك بسبب شفافة مناطق تشكيل ذات المستعمرات وقد تم اختيار .االجار

انتاجأكبر أظهرت ساللة واحدة قد وجد أن .ةدرجة مئوي 4 حفظ السالالت البكتيرية بعد تنقيتها فى

درجة مئوية 45 و 33 بالمقارنة ب درجة مئوية 33 ان درجة الحرارة المثلى هى لوحظ. نزيملال

عصوية نهاا بصبغ السالله الناتجة النزيم البروتييز بصبغة كريستال فايلوت وجد. على التوالي

موجبة الصبغة سالالت بكتيرية باستخدام زييالبروت انزيمإنتاج ,عامة. الشكل و موجبة صبغة جرام

و تطرية األمينية واألحماض، الذائبة اتبروتينال صناعة مثل ةيدتشجع قيام صناعات عدجرام

معالجة المخلفات و ، الجلوددباغة صناعة األدوية، و ,مساحيق الغسيلصناعة األلبان،و اللحوم

. مفيدة إلى مركبات تحويلها

20

المملكة العربية السعودية

كلية العلوم -جامعة جازان

قسم االحياء

ة جازانعزل ساللة بكتيرية منتجة النزيم البروتييز من منطق

و دراسة تأثير درجة الحرارة على انتاج االنزيم

اعداد الطالب

اسماعيل حسن احمد ملحان

810100102:رقم جامعى قسم األحياء -ئيةطالب السنه النها

جامعة جازان –كلية العلوم

اشراف

خالد السيد الجيار/د قسم االحياء –استاذ مساعد

جامعة جازان -كلية العلوم

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