Protease An Enzyme Capable To Hydrolyze Proteins Biology Essay

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Protease is an enzyme that capable to hydrolyze proteins to short peptides or free amino acids. This enzyme has a wide range of application, including in food, detergent, pharmaceutical, leather, and chemical industries (Huang et al., 2006). Generally, enzyme is used in industry as potential biocatalyst for a lot of reactions (Ibrahim, 2007).

According to the Ferrero et al. (1996), commercial proteases are typically produced from various bacteria sources. This is due to higher quantities of enzyme produced by the microorganisms rather than plant or animal sources (Ibrahim, 2007). In addition, there is a demand for thermostable proteases from a few industries for their superior heat tolerance that allowed them to function at high processing temperature (Nascimento and Martins, 2004). Therefore, thermophile which is an organisms that capable of living at high temperature between 45 to >70 °C (Nazina et al., 2001) can be one of the sources for producing thermostable proteases as the enzymes secreted by thermophile were reported to be able to withstood heat at higher degree compared to the enzymes secreted by mesophiles (Salem et al., 2009).

Fermentation process had been recognized as a great method for production of enzyme from microbial sources. Nowadays, production of enzyme from microorganisms can be carried out by fermentation process using cheap substrate and production systems (Ibrahim, 2007). Therefore, fermentation by using flask technique was preferably to produce protease from bacterial sources within the laboratory scale. Crude protease extract have been tested on skim milk agar assay and litmus milk assay in the way to determine an efficiency of fermentation process to produce the desired enzyme.

PROBLEM STATEMENT

Production of enzymes and their applications are also one of the high potential economic resources. Developed country like Japan has been successfully produced enzyme on commercial scale. However, Malaysian enzyme industry is still considered lacking or almost absent of effort to develop commercial scale of enzyme production although our country is rich in natural sources for enzyme producers. Thus, preliminary study on fermentation by using natural source such as local thermophile should be done to encourage the initial effort of developing large scale Malaysian enzyme industry.

SIGNIFICANT OF STUDY

This study embarks on fermentation of thermophilic bacteria by flask technique to produce thermostable proteases will serve as an initiation step to promote local enzyme productions. Flask fermentation technique was shown to be appropriate and easy method to express sufficient volumes of enzymes which can facilitate further study on characteristic of this unique enzyme.

OBJECTIVES

1.4.1 General objective

To ferment thermophile A8 to produce thermostable protease by using flask technique.

1.4.2 Specific objectives

To maintain thermophile A8 in laboratory.

To ferment thermophile A8 in order to induce protease production.

To perform extraction of protease from the fermented thermophile A8.

To prove the presence of protease in the crude enzyme extract.

HYPOTHESIS

Ha : Protease can be produced by fermentation of thermophile A8 using flask technique.

CHAPTER 2

LITERATURE REVIEW

2.1 PROTEASE ENZYME

Protease is an enzyme which belongs to the class of enzymes known as hydrolases which function to breakdown proteins. This enzyme will catalyzes large number of reaction by hydrolyses the peptide bonds that link amino acid chains that make up protein molecules (Chaplin and Bucke, 2004). Protease applications have been seen in leather processing, brewing and food industries but the most important of its applications are used in laundry detergents (Kebabci and Cihangir, 2011). Other enzymes including protease that used within industrial applications are shown in the Table 2.1.

Table 2.1: The uses of enzymes in industrial application (Kirk et al., 2002).

Application

Enzymes used

Uses

Food processing

Amylases

Production of sugars from starch, such as high-fructose corn syrup.

Proteases

To lower the protein level of flour in biscuit manufacturing.

Brewing industry

Amylase, glucanases, and proteases

To split polysaccharides and proteins in the malt.

Proteases

To remove cloudiness that produced during storage of beers.

Dairy industry

Rennin (derived from the stomach of young ruminant animals like calves)

To hydrolyze protein in manufacturing of cheese.

Lipases

Implemented during the production of Roquefort cheese to enhance the ripening of the blue-mould cheese.

Lactases

Break down lactose to glucose and galactose.

Meat tenderizers

Papain

To soften meat for cooking.

Biological detergent

Primarily proteases, produced in an extracellular form from bacteria

Used for pre-soak conditions and direct liquid applications helping with removal of protein stains from clothes.

Amylase

Detergents for machine dish washing to remove resistant starch residues.

Lipases

Assist in the removal of fatty and oily stains.

Cellulases

Used in biological fabric conditioners

Contact lens cleaners

Proteases

Remove proteins on contact lens to prevent infections.

Rubber industry

Catalase

Generate oxygen from peroxide to convert latex into foam rubber.

Molecular biology

Restriction enzymes, DNA ligase and polymerases

Manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine. Essential for restriction digestion and the polymerase chain reaction.

2.2 VARIOUS SOURCE OF PROTEASE ENZYME

Protease represented as one of the most important groups of industrial enzymes and cover about 60% of total enzyme market as reported by Nunes et al. (2001). Plants, animals and microbes had been recognized as the main sources for protease production. However, microbial proteases are the most significant sources rather than animal and plant protease. This is due to the characteristic of the microorganism itself where it can growth rapidly, only required limited space for cultivation, capable to utilize low-cost nutrients, and it also can be genetically manipulated to produce new enzymes with altered properties (Usharani and Muthuraj, 2010). Several commercial enzymes from microbial sources were showed in the Table 2.2. Among bacteria sources, Bacillus sp. is potential in production of these extracellular enzymes under fermentation conditions (Salem et al., 2009).

Table 2.2: Enzymes from microbial sources that isolated from Malaysian soils (Ibrahim, 2007).

Enzyme

Microorganism

Lipase

Aspergillus oryzae, Aspergillus flavus, Mucor miehei, , Bacillus sp., Corynebacterium sp., Geobacillus thermodenitrificans

Protease

Bacillus subtilis, Bacillus megaterium, Trichoderma sp., Aspergillus niger

Cellulase, β-glucosidase

Aspergillus niger, Trichoderma reesei

Xylanase, Tannase and Mannanase

Aspergillus niger

Bio-Catalysts Isolated From Malaysian Micro-Organisms

Type of Bio-Catalyst Micro-Organism

2.3 THERMOPHILIC BACTERIA

Thermophilic bacteria are defined as organisms that capable of living and growing at high temperature between 45 to >70 °C (Nazina et al., 2001). These bacteria not only survive but are able to thrive in boiling water. Therefore, it is commonly found in various geothermal heated regions such as hot springs (Elnasser et al., 2007).

Previous studies have been deal with the question how thermophilic bacteria can survive at high temperatures (Nordstrom, 1993). This is because the bacteria cells are surrounded by a membrane lipid which plays an important role in the fluidity of membranes. Thermophiles will modify the phospholipids compositions which maintain both the membrane integrity and fluidity in order to adapt the extreme temperature (Nordstrom, 1993).

Besides that, some studies were performed to isolate thermophilic bacilli, including Geobacillus from all geothermal areas (McMullan et al., 2004), intent on the use of these organism and its enzymes for biotechnological application. Geobacillus refers to 'earth' or 'soil' Bacillus (Nazina et al., 2001), where thermophilic bacteria that belonging to Bacillus genetic group 5 have been reclassified as being members of this genera. As with many thermophiles, Geobacillus and their gene products seems significance in industrial application although these bacteria may be a newly described genus (McMullan et al., 2004).

2.4 THERMOPHILIC BACTERIA AS SOURCE OF THERMOSTABLE PROTEASE

Thermophilic bacteria had been found as a source for producing thermostable enzyme since this organism can survive and secreted enzyme at high temperature. Bacillus sp. was recognized as thermophilic bacteria which become good producers of extracellular thermostable protease. Thermophilic Bacillus sp. such as Bacillus stearothermophilus (Boonyanas et al., 2000), Bacillus pumilus (Kumar, 2002) and Thermophilic Bacillus strain HS08 (Huang et al., 2006) have been studied for their potential in producing thermostable protease. The demand on the thermostable enzyme rises due to its important in some commercial uses such as for PCR reaction in molecular biology application. These unique enzymes are advantageous because of its ability to withstand intense heat and also provide a high activity and faster reaction rates (Adams et al., 1998). In addition, because of its beneficial, thermophilic protease can also be used as ideal models for studying thermal stability of protein (Huang et al., 2006).

2.5 SHAKE FLASK FERMENTATION

In its broadest area, fermentation means any process by which large organic molecules are broken down to simpler molecules as the result of the action of microorganisms. Such an example, conversion of sugars and starches to alcohol by enzymes in yeast have become the most familiar type of fermentation process (Bajaj and Sharma, 2011). In this study, flask fermentation technique refers to the process production of enzymes by growing the microorganisms that produce the product in Erlenmeyer flask. Although the process is only done in flask, but it has mimic the truly large scale fermentation process which equivalent to stirred tank bioreactor.

In general, there are two types of fermentation that can carried out by flask technique which are submerged fermentation and solid-state fermentation, and proteases had been found that can be produced by both types of fermentation (Chutmanop et al., 2008). However, submerged fermentation is preferred to use in this study where fermentation process was performed by cultivation of the microorganism that produced the enzyme in a fermentation medium. Furthermore, the use of flask fermentation technique is advantageous because of the ease of sterilization and process control and also provides special interest in those processes where the crude fermented product may be used directly as the enzyme source (Vidyalakshmi et al., 2009).

Figure 2.1 showed the flow for the preparation of enzymes from different sources. However, the aim of this study only focuses for the microbial fermentation to produce extracellular enzymes. In this study, centrifugation is the preferred method to concentrate the fermented medium and clear supernatant of the solution was used as crude enzyme extract (Chaplin and Bucke, 1990).

Figure 2.1: Flow diagram for the preparation of enzymes from different source (Chaplin and Bucke, 1990).

2.6 DETERMINATION ON PRESENCE OF PROTEASE IN CRUDE ENZYME EXTRACT

Determination of fermentation and extraction efficiency is based on presence of protease in the crude enzyme extract. Method used for that purpose is SMA assay, and litmus milk assay since proteases have potency to hydrolyze several protein substrates (Huang et al., 2006). Each principle of the assay is described as follows:

2.6.1 Skim Milk Agar Assay

This assay is used as a direct technique to identify the presence of protease enzyme in the crude extract. Skim milk agar is prepared for that purpose. Presence of protease in the crude extract can be reveal based on its ability to enzymatically degrade proteins to peptones (peptonization). The result of peptonization reaction was shown by formation of lytic zone on the skim milk agar (Downes and Ito, 2001).

2.6.2 Litmus Milk Reaction

This assay is used as an indirect technique to identify the presence of protease enzyme in the crude extract. Generally, when the litmus milk is added with distilled water, the milk suspension is turns to purple color. Litmus milk medium consists of several components such lactose, casein and the dye molecule litmus which can be metabolized by different organism. Litmus acts as a pH indicator and also as a reducible dye molecule. In an acid condition, litmus will turns from purple to pink color, while from purple to pale blue in alkaline environment. Litmus milk reaction can aid in the determination on presence of protease because action of protease on protein in milk results in the release of ammonia and lead to an alkaline condition which turns the litmus to pale blue. (Cheesbrough, 2009, Downes and Ito, 2001).

CHAPTER 3

MATERIALS AND METHODS

Maintain thermophile A8 in laboratory

Preliminary identification of thermophile A8

(Gram stain, motility test)

Fermentation of thermophile A8 by flask technique

Extraction and filtration of crude enzyme

Litmus milk assay

Skim milk assay

Figure 3.1: Flow chart describing the overall processes of this study

3.1 BACTERIA STRAIN

In this study, one thermophilic bacteria sample have been used which provided by postgraduate student. The sample was previously isolated from the hot spring at Sg. Klah, Perak and coded as A8 sample (Andrew, 2008). Thermophile A8 has been found to produce protease from the previous study.

3.2 MAINTENANCE OF THERMOPHILE A8

3.2.1 Preparation of Culture Media

Castenholz Tryptone Yeast Extract (CTYE) medium was prepared according to the method from previous study (Andrew, 2008). Composition of Castenholz 2x basal salts stock contains (per liter of distilled water): nitrilotriacetic acid, 0.2g; calcium sulfate, 0.12g; magnesium sulfate, 0.2g; sodium chloride, 0.016g; potassium nitrate, 0.21g; , sodium nitrate, 1.4g; sodium phosphate, 0.22g; ferric (III) chloride solution, 2ml; and Nitsch's Trace Element Solution, 2ml. Nitsch's Trace Element Solution contains (per liter of distilled water): sulphuric acid, 0.5ml; manganese sulfate 2.2g; zink sulfate, 0.5g; boric acid, 0.5g; copper (IV) sulfate, 0.016g; sodium molybdenum, 0.025g; and cobalt chloride, 0.046g. The pH of Castenholz 2x basal salts stock was adjusted to 8.2 with 1.0 M NaOH and this basal medium was sterilized by autoclaving at 121oC for 15 min.

Stock solution of 1% tryptone yeast extract (TYE) was prepared by dissolved 5g of tryptone and 5g of yeast extract in 500 ml distilled water and then stock solution was sterilized by using autoclave (Hirayama, Japan) at 121oC for 15 min. The complete 1L of CTYE broth was made by aseptically mixing five parts of double strength Castenholz salts with one part of 1% TYE and four parts of distilled water which makes the final concentration of TYE is 0.1%.

CTYE agar was prepared by adding 30g of Bacto agar powder (BD, USA) into 1L of CTYE broth. So that, the CTYE agar concentrations is 3%. Agar medium was autoclaved at 121oC for 15 min and then poured aseptically into disposable petri dishes.

3.2.2 Culture Condition

CTYE agar was used to subculture thermophile A8 that obtain from previous study. A8 subculture plate was incubated at 60oC for 24h in the incubator. CTYE broth also used to cultivate the bacteria sample. 10 ml of CTYE broth was poured into a 100 ml Erlenmeyer flask equipped with cotton-wool stoppers. Then, three to four colonies of thermophile A8 from 24h culture was inoculated into the broth. Flasks containing broth culture was incubated in shaking water bath (Memmert, Germany) at 60oC and was observed up to two days of incubation period. Morphology of the bacteria colony on CTYE agar was observed after 24h incubation, while any changes of color and turbidity of bacteria broth culture was observed and record after two days incubation.

3.3 PRELIMINARY IDENTIFICATION OF THERMOPHILE A8

3.3.1 Gram Stain

A smear of single colony bacteria from 24h culture was done on the glass slide. The smear was flooded with a crystal violet solution for one minute. After one minute, the slide was washed with running tap water and then iodine solution was added to the smear for one minute. After that, the slide was washed with running tap water again and decolorized rapidly with alcohol. The alcohol was washed up with tap water and the slide was counterstained with safranin for one minute. After the slide being washed with tap water, let it to dry. The shape and gram reaction of the bacteria was observed under a light microscope by using 100X oil immersion lens.

Positive and negative control for gram-stain was done together in this process to validate the result and observation. Staphylococcus aureus is used as a Gram-positive control while Escherichia coli are used as Gram-negative control.

3.3.2 Motility test (Hanging Drop Method)

Hanging drop method for motility test was performed by making a round shape using plasticin on the glass slide. A bacteria colony was inoculated in saline on the cover slip. Then, glass slide was placed on the cover slip and turn back the glass slide. So that, the bacteria was hanging on the cover slip. The movement of the bacteria was observed under a light microscope by using 40X lens.

3.4 FERMENTATION OF THERMOPHILE A8 BY FLASK TECHNIQUE

Protease production by flask fermentation technique was performed according to method of Nascimento and Martins (2004) with some modification. Fermentation by flask technique was initiated by inoculation of three to four colonies of 24 h old thermophile A8 into the fermentation medium in 250ml Erlenmeyer flask equipped with cotton-wool stoppers. Fermentation medium consists of 15 ml skim milk solution and 85 ml CTYE broth. Skim milk and CTYE broth are used as substrate to induce the production of protease in the fermentation process. After inoculation of bacteria sample, fermentation by flask technique was carried out at 60oC in shaking water bath up to two days of incubation period.

3.5 EXTRACTION AND FILTRATION OF CRUDE ENZYME

Extraction of crude enzyme was performed according to method by Das and Prasad (2010) with some modification. After each fermentation period, the whole ferment medium was centrifuged at 5000 rpm for 30 min at 4oC to remove the cellular debris and the clear supernatant was used as crude enzyme source. Sterility for the crude enzyme source was done by filtration of the clear supernatant with 0.2µm syringe filter to eliminate other organism in the crude enzyme extract. Then, crude enzyme extract was stored at 4oC refrigerator until for the use.

3.6 DETERMINATION ON PRESENCE OF PROTEASE IN CRUDE ENZYME EXTRACT

3.6.1 Skim Milk Agar Assay

The ability of enzyme extract to hydrolyze protein was tested by SMA assay. SMA was prepared by dissolve 10g of skim milk powder (BD, USA) and 20g Bacto agar powder in 500 ml of distilled water. Agar medium was sterilized by autoclaving at 121oC for 15 min. Then, SMA was poured aseptically into disposable petri dishes. To perform the SMA assay, 20µl of crude enzyme extract was pipette and placed on the plain antibiotic disc located on the SMA. Ensure that enzyme extract do not contaminate with the SMA during the procedure. Then, the agar plate was incubated 24h at 60oC in incubator.

Enzyme extract was put on the plain disc

Plain antibiotic disc

SMA

Figure 3.2: Procedure of SMA assay for observation zone of lysis on SMA done by protease enzyme.

3.6.2 Litmus Milk Assay

Litmus milk medium was prepared by dissolve 50g of litmus milk powder in 500 ml of distilled water. Medium was autoclaved at 121oC for 15 min. Then, 5 ml of crude enzyme extract was poured into test tube containing 10 ml of litmus milk medium. The mixture is stirred on a vortex mixer for five seconds. After vortex, test tube was incubated in shaking water bath at 60oC and up to three days of incubation period. One tube with 10 ml of litmus milk medium without enzyme extract was done together as a negative control.

CHAPTER 4

RESULTS

4.1 MAINTENANCE AND PRELIMINARY IDENTIFICATION OF THERMOPHILE A8

After 24h cultivation of thermophile A8 on CTYE agar at 60oC, the result showed the presence of single creamy colony on the agar. The morphology of the colony describes as having small size of colony, circular shape, entire margin and convex elevation of the colony. For the thermophile A8 broth cultivation at 60oC, the result showed the turbidity of CTYE broth culture after two days of incubation period compared to the original color of the CTYE broth. Besides that, result for broth culture also indicates the presence of whitish precipitate at the bottom of the flask.

Gram-stain provide as a preliminary identification of the thermophilic bacteria. Result for the gram-stain of single colony thermophile A8 showed that the bacterium was Gram-negative bacilli with spore forming. This result was validated by comparing the Gram-stain color of the thermophile A8 against laboratory strains of Staphylococcus aureus and Escherichia coli as Gram-positive and Gram-negative controls respectively. Hanging drop method for motility test revealed that thermophile A8 is motile when observed under 1000X magnification using light microscope.

Single creamy colony of thermophile A8

A8 21/2

Plate 4.1: Presence of single creamy colony of thermophile A8 on the CTYE agar after 24h incubation at 60oC.

Spore

Plate 4.2: Gram-negative bacilli with spore forming of thermophile A8 observed under 1000X magnification using light microscope.

A

B

Plate 4.3: Control for gram-stain. A: Gram-positive control (Staphylococcus aureus) shows dark purple color of bacteria. B: Gram-negative control (Escherichia coli) shows red color of bacteria.

4.2 EXTRACTION OF CRUDE ENZYME FROM THERMOPHILE A8 FERMENTATION

Fermentation of thermophile A8 in the flask after two days of incubation at 60oC was showed the changes in appearance of fermentation medium from milky white to clear brown color. Crude enzyme was successfully extracted from the fermented medium by using centrifugation method where the clear supernatant serves as an enzyme sources. 90 ml of clear supernatant was successfully extracted from fermentation medium and used as a crude enzyme source. Sterility of the crude enzyme extract represent that the filter crude enzyme does not possess bacterial growth on CTYE agar while non-filter crude enzyme showed the numerous growths of bacteria on the same agar. Therefore, filter crude enzyme preferably used in the qualitative assay for determination on presence of protease in the crude enzyme extract.

A8

Clear brown color of fermentation medium

Plate 4.4: Clear brown color of fermentation medium after two days of incubation at 60oC compare to its original milky white color.

4.3 DETERMINATION ON PRESENCE OF PROTEASE IN THE CRUDE ENZYME EXTRACT

4.3.1 Skim Milk Agar Assay

Crude enzyme extract shows positive result on SMA after 24 h incubation at 60oC. Protease presence in crude enzyme was capable to hydrolyse protein in skim milk and the formation zone of lysis on SMA was shown in Plate 4.5.

Lysis zone on skim milk agar

Plate 4.5: Zone of lysis on skim milk agar indicates the proteolytic activity by protease in enzyme extract.

4.3.2 Litmus Milk Assay

Reaction on litmus milk was observed after three days upon incubation at 60oC. Enzyme extract shows positive result on litmus milk assay which indicated by the changes color of litmus milk solution from purple to pale blue color.

B

A

Plate 4.6: Changes in litmus milk color from (A) purple to (B) pale blue color after three days of incubation at 60oC.

CHAPTER 5

DISCUSSIONS

5.1 MAINTENANCE OF THERMOPHILE A8 IN LABORATORY

From this study, thermophile A8 was successfully have been cultured on Castenholz Tryptone Yeast Extract (CTYE) agar and maintained in the laboratory at 60oC. The 24h culture at optimum temperature have effectively produced creamy single colony of thermophilic bacteria A8 on the CTYE agar. The morphology of the colony on agar plate represents a small size of colony, circular shape, entire margin and convex elevation of the colony. This result revealed that CTYE agar as a medium that can support the growth and maintaining of the bacteria at high temperature. Since thermophile A8 can growth efficiently on the CTYE agar, CTYE broth also hopefully can support the growth of bacteria in the broth culture. As CTYE contains sufficient nutritional requirement in that solution such as tryptone, yeast axtract and some trace elements, it can also be use as fermentation medium as the carbon, nitrogen and metal ions inside this medium were to be able to induce production of bacterial extracellular protease.

5.2 INDEFINITIVE GRAM-STAIN PROFILE OF THERMOPHILE A8

Preliminary identification of thermophilic bacteria A8 represent that these organisms recognize as Gram-negative bacilli with spore forming and motile. In this study, control for gram-stain was done using Gram-positive and Gram-negative known organism in order to prevent misinterpretation of bacteria gram-reaction. The result indicates that a bacterium is Gram-negative because the color is quite similar to the gram-stain of negative control. However, based on a few available literatures, thermophilic bacilli sp. has been establish as Gram-positive bacteria. Geobacillus sp. was reported to possess cell wall structure resembling the Gram-positive bacteria (Nazina et al., 2001). However, variable Gram-stain profiles have been observed by many literatures such as Banat et al., (2004), which report that Geobacillus debilis organism stained Gram-negative while Gram-positive Geobacillus thermodenitrificans organism have been observed by Liu et al., (2008). Moreover, the result also may expect due to performing the Gram-stain during the thermophile's stationary phase culture of bacteria. The reason is because many spore-forming bacteria were rapidly become Gram-negative when entering this growth phase as reported by Todar (2004). In order to overcome this, it is best to perform the Gram-stain at the exponential growth phase as this is the optimum period to perform any biochemical testing on bacteria (Tortora et al, 2004). Motility of this organism are proven because almost all species of Bacillus are motile except for the most virulent, which is Bacillus anthracis (Cheesbrough, 2009).

5.3 PROTEASE PRODUCTION IN FLASK FERMENTATION TECHNIQUE

Proteases production was successfully conducted using fermentation by flask technique which constitute total of 100 ml fermentation medium and inoculated thermophile A8. The result showed that fermentation medium turns to clear brown color after two days of incubation at 60oC compare to its original milky white color. The color changes have proved that fermentation process was effectively occurred and proteases enzyme have been produced within the process. The result may also indicated that skim milk as the suitable fermentation substrate used in this process where protein in milk have been subjected to be hydrolyse by the protease produced by the bacteria. This result agree with the studies by Chutmanop et al. (2008), where substrate with high protein content can stimulate enzyme production, and results in highest proteases activity. Based on researcher observation, the fermentation process in this study demonstrate that large protein molecule in skim milk are broken down to simpler molecules where the conversion reaction is catalyzes by the proteases enzyme that produced by the bacteria and demonstrated by clearing of the creamy white solution of the fermentation medium to clear brown color after two days of fermentation process at 60oC.

Extraction of the fermentation product becomes a great challenge since the requirement to concentrate the fermented broth was found to be complex. According to method by Olajuyigbe and Ajele (2008), the whole fermented broth need to be centrifuged at 10,000 rpm for 15 min at 4oC using refrigerated centrifuged and then supernatant was used as crude enzyme source. However, result in this study represent that the 90 ml of crude protease extract after centrifugation with modification seem as diluted and there is difficult to determine the actual concentration of proteases obtained in the crude extract. This is because unable to get the desired spin speed of the centrifuge to concentrate the fermented medium.

Filtration of the crude enzyme extract was done in order to sterilize it. So that, the sterile crude enzyme extract can be use for the qualitative assay in determination presence of protease in that extract. Filtration of the extract can ensure that no bacterial is present in the extract, only the desired enzyme suppose available in that extract. This is because, hopefully that enzyme extract can cause the positive result on the assay, not the bacterial that possess in that extract. Other than that, sterilization of the enzyme extract by filtration method was done due to enzyme cannot be autoclaved or chemically steril because enzyme will be denatured by that process.

Moreover, flask fermentation will provide a good starting point for better large scale production of local enzyme in industrial. This is because optimization of fermentation condition such as type of substrate used, substrate volume, and incubation temperature can be done prior in small scale before the process will proceed in the large scale fermentation. In addition, fermentation by flask technique only required small volume of substrate or reagent rather than fermentation in bioreactor (Chutmanop et al., 2008).

5.4 DETERMINATION ON PRESENCE OF PROTEASE IN THE CRUDE ENZYME EXTRACT

Based on the results of SMA assay, have been proved that the thermophile A8 is thermostable proteases producing strain since it's crude enzyme extract provide significantly active proteolytic activity on SMA at high temperature, which showed by the presence clear zone of lysis around the plain antibiotic disc on SMA after 24h incubation period at 60oC. These results support the earlier findings by Usharani and Muthuraj, (2010) which found that protease produce by Bacillus laterosporus strain capable to hydrolyse protein in the skim milk and forming the clear lyses zone on SMA.

Besides SMA assay, litmus milk assay also yields the positive result towards determine presence of protease in crude enzyme source. Positive results for this assay represent by the changes of litmus milk color from purple to pale blue color after three days incubation period at 60oC. This result has been proved the enzyme extract as protease since its proteolytic activity towards protein in milk resulting in the release of ammonia and lead to an alkaline condition which turns the litmus color.

CHAPTER 6

CONCLUSION AND RECOMMENDATION

The main purpose of this study is to ferment thermophilic bacteria A8 to produce thermostable proteases by using a flask technique. Therefore, based on the results obtained, it can be concluded that thermostable proteases have been successfully produced by fermentation of thermophile A8 using flask technique at 60oC. In addition, determination of the fermentation and extraction efficiency proved that the crude enzyme extract is belongs to proteases since its potency to form clear lysis zone on skim milk agar and provide changes in litmus milk color from purple to pale blue color. These positive results are due to the ability of proteases to hydrolyzed protein in milk for the both qualitative assay.

In this study, purification of thermostable proteases was not performed. Hence, further studies are needed to find out the proper way to concentrate and purify the proteases extract in order to get the desired crude proteases source. In conclusion, the objectives of this study were achieved, and the results have support the alternative hypothesis. Proteases can be produced by flask fermentation technique of thermophile A8.

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