Proteases Are A Group Of Enzymes

Published Date: 02 Nov 2017

CHAPTER 1

INTRODUCTION

Proteases are a group of enzymes, which responsible for hydrolysis peptide bonds of proteins and break them down into smaller polypeptides or free amino acids (Gupta et al., 2005). Proteases execute a large variety of functions which find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes (R.Gupta et al., 2002). Proteases represent one of the three largest groups of industrial enzymes and account for about 59% (Fig. 1) of the total worldwide sale of enzymes (Godfrey and West., 1996).

Analytical and Pharmaceutical enzyme

Alkaline protease

Other

proteases

Trypsin

Lipases

Carbohydrases

Amylases

Rennins

Figure 1.1: Distribution of enzyme sales. The contribution of different enzymes to the total sale of enzymes is indicated. The yellow portion indicates the total sale of proteases.

Proteases are widely distributed nearly in all plants, animals and microorganisms which participate in a variety of metabolic and regulatory functions in all organisms. However, microbial protease is a main source of industrial enzyme due to the inability of the plant and animal proteases to meet current world demands. Microbes represent an attractive source as they can be cultured in large quantities in a relatively short time, the limited space is required for their cultivation and ease of genetic manipulation (Josephine et al., 2012).

Microorganisms can secrete intracellular and/or extracellular protease. Intracellular proteases are important for various cellular and metabolic processes, such as sporulation and differentiation, maturation of enzymes and hormones, protein turnover, and maintenance of the cellular protein pool whereas extracellular proteases are important for the hydrolysis of proteins in cell-free environments and enable the cell to absorb and utilize hydrolytic products (Kalisz 1988). All of the predominant industrial microbial proteases are secreted as extracellular enzymes into the culture medium. Most commercial proteases are produced by Bacillus and Aspergillus species due to their high growth rates and short fermentation durations (Ward, 2011).

Production of proteases enzyme has attracted the attention of many researchers, as these enzymes have wide range of application. Looking into the depth of microbial diversity, scientists prefer studying on new isolates because they could be alternative for commercial use. There is always a chance of finding microorganisms producing novel enzymes with novel properties and suitable for commercial exploitation. Taking into this consideration, a research to isolate newer source of protease producing bacteria from the local soil and water sample was carried out. There is a need to find microbial enzymes that exhibit high proteolytic activity at wide range conditions and can be produced economically. The objectives of this study include:

To screen and select protease-producing bacteria from soil and water samples

To identify and characterize protease producing microbes

To determine the optimum temperature, pH and carbon sources on proteasee production of the selected strains

Protease-producing bacteria were screened and isolated from soil and water sample. Phenotypic and genomic analyses were achieved using biochemical tests and the gene sequence analysis amplified by polymerase chain reaction (PCR) technique. Factors affecting growth and protease production of the selected strains were studied, which included temperature, pH and carbon sources.

CHAPTER 2

LITERATURE REVIEW

2.1 Classification of microbial protease

Proteases represent a large and diverse group of hydrolytic enzymes which can be classified in a number of ways, for example, on the basis of their site of action, or their functional group at active site, or their catalytic mechanism.

Depending on their site of action, protease can be subdivided into two major groups which are exopeptidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate whereas endopeptidases cleave peptide bonds distant from the termini of the substrate (Hartley 1960). Based on the catalytic mechanism , proteases are further classified into six prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases, metalloproteases, glutamic acid protease and threonine protease(O.P.Ward et al., 2009).

2.1.1 Exopeptidases

The exopeptidases act only near the ends of polypeptide chains and are classified as aminopeptidases and carboxypeptidase based on their site of action at the N or C terminus (O.P.Ward et al., 2009).

Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide, or a tripeptide (Fig 2.1). Aminopeptidases occur in a wide variety of microbial species including bacteria and fungi (Watson 1976). In general, aminopeptidases are intracellular enzymes, but there has been a single report on an extracellular aminopeptidase produced by Aspergillus oryzae(O.P.Ward et al., 2009).

The carboxypeptidase act at C terminals of the polypeptide chain and liberate a single amino acid or a dipeptide (Fig2.1). Carboxyproteases are characteristically differentiated based on amino acid substituent at the active site into three major groups which are serine carboxyproteases, metallocarboxyproteases, and cysteine carboxyproteases (Rao et al., 1998).

Figure 2.1: Classes and active site of exopeptidases.Open circles represent the amino acid residues in the polypeptide chain. Solid circles indicate the terminal amino acids. Solid triangle indicates site of peptide cleavage.

2.1.2 Endopeptidases

Endopeptidases are characterized by attacking peptide bonds in more central locations of the polypeptide chain away from the N and C termini and free amino or carboxyl groups that are known to inhibit or retard enzyme action. They are divided into six subgroups based on their catalytic mechanism(O.P.Ward et al., 2009).

2.1.2.1 Cysteine protease

Cysteine proteases occur in both prokaryotes and eukaryotes. Based on their side chain specificity, cysteine proteases broadly divided into four group: (1) papain-like, (2) trypsin-like with preference for cleavage at the arginine residue, (3) specific to glutamic acid, and (4) others. Most of the cysteine proteases have neutral pH optima.(Rao et al., 1998). All cysteine proteases have cysteine/histidine catalytic dyad and the order of these residues differs among the families ( Barett 1994). Reducing agents such as sodium bisulfite, hydrogen cyanide, or cysteine are needed by cysteine protease for activity retention.

Clostripain produced by Clostridium histolyticum and Streptopain produced by Streptococcus Spp are the example of cystein protease (Rao et al., 1998).

Aspartic protease

Most of the aspartic proteases exhibit maximal activity at low pH, therefore, they are known as acidic proteases (O.P.Ward et al., 2009). The active-site aspartic acid residue is situated within the motif Asp-Xaa-Gly, in which Xaa can be Serine or Threonine. The activity of microbial aspartic proteases similar to pepsin which show specificity against aromatic or bulky amino acid residues on both sides of the peptide bond, but their action is less stringent than that of pepsin. Microbial aspartic protease are divided into two groups which are (1) pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora; and (2) rennin-like enzymes produced by Endothia and Mucor spp(Rao et al., 1998).

Serine protease

Serine proteases are characterized by containing a conserved glycine-containing peptide, Gly- Xaa-Ser-Yaa-Gly, associated with the catalytic serine which is binding site of substrate. essential for substrate (O.P.Ward et al., 2009). They are numerous and widespread among viruses, bacteria, and eukaryotes which help to catalyze the hydrolysis of esters and amides as well as peptides(Rao et al., 1998).Among the families of serine protease, the important well-known enzymes of serine protease include the chymotrypsins and the subtilisins. The subtilisins are an important family of serine proteases produced by Bacillus species (Ward, 2011). Serine proteases are generally active at neutral and alkaline pH, with an optimum between pH 7 and 11(Rao et al., 1998).

2.1.2.4 Metalloprotease

The activity of metalloproteases require the present of a divalent metal ion. The important well-known enzyme of metalloprotease is thermolysin which is produced by B. stearothermophilus. The active site of Thermolysin consist of histidine and glutamine which providing a ligand for zinc and a catalytic function, respectively. (O.P.Ward et al., 2009)

Metalloproteases can be sub-classified into four groups according the specificity of their action which are (i) neutral, (ii) alkaline, (iii) Myxobacter I, and (iv) Myxobacter II. The neutral proteases exhibit specificity for hydrophobic amino acids whereas the alkaline proteases have a very broad specificity. Myxobacter protease I is specific for small amino acid residues on either side of the cleavage bond, whereas protease II is specific for lysine residue on the amino side of the peptide bond(Rao et al., 1998).

2.1.2.5 Threonine protease

Threonine protease is a new class of enzyme which was discovered in 1995 as part of proteasome complex. The discovery of this protease is based on the observation of its crystallographic structure of the eukaryotic proteasome (XT01-001) from Saccharomyces cerevisiae .An N-terminal threonine is found at the active site. The ability of proteasome to resist most standard protease inhibitors discovered the catalytic function.(O.P.Ward et al., 2009)

2.1.2.6 Glutamic acid protease

Glutamic acid protease is new enzyme family which was derived from the former pepstatin insensitive carboxyl proteases. Glutamic acid is found to be insensitive to pepstatin. Glutamic acid and glutamine in active site play an important role in binding of substrate and catalysis.These enzymes have been discovered from Stylidium lignicola and Aspergillus niger var. macrospores (O.P.Ward et al., 2009)

2.2 Commercial Applications of Protease

Microbial proteases constitute a complex range of enzymes with wide spectrum of properties that make them suitable for different commercial applications.

2.2.1 Detergent

The use of proteases in laundry detergents accounts for approximately 25% of the total worldwide sales of enzymes. Detergent proteases have long been of interest to the detergent industry for their ability to remove large verity of stain due to food, blood, and other body secretions. All detergent proteases currently used in the market are serine proteases produced by Bacillus strains. (Rao et al., 1998)

The evaluation of detergent proteases is mainly dependent upon parameters such as the pH and ionic strength of the detergent solution, the washing temperature and pH, mechanical handling, level of soiling and the type of textile. The protease perform well when its pI conincides with the pH value of the detergent solution (R.Gupta et al., 2002).

Current market trends and consumer demands are influencing the development of detergent protease in detergent industry. Therefore, there is the need to find newer enzyme to enhance the wash performance of currently used enzyme-based detergents. Recently, the proteases which are active at lower temperature are needed in detergent industry due to the energy crisis and the awareness for energy conservation(Rao et al., 1998). Besides that, the increased use of synthetic fibers which cannot tolerate high temperature had pushed the enzyme manufacture to find novel enzyme that can act under low temperatures (Hasan and Tamiya 1997; Kitayama 1992; Nielsen et al. 1981).

2.2.2 Leather Industry

Leather processing consists of soaking, dehairing, bating, and tanning. The use of hydrogen sulfide and other hazardous chemicals in conventional method of leather processing had caused the environmental pollution. Thus, the use of enzymes instead of chemicals is an alternative ways to solve the pollution problems and improve the leather quality (Andersen 1998).

In chemical method for dehairing, the need of creating extremely alkaline conditions followed by treatment with hydrogen sulfide to removes the proteins of the hair root. At present, alkaline proteases with hydrated lime and sodium chloride are used in order to reduce the time required in dehairing process and prevent the pollution problems caused by hazardous compound..(Rao et al., 1998).

For earlier bating methods, animal feces was used as the sources of proteases. At present, the use of pancreatic trypsin has replaced this unpleasant and unreliable method. Trypsin is used in combination with other Bacillus and Aspergillus proteases for bating. The consideration of protease selection is mainly depend upon the its specificity for matrix proteins such as elastin and keratin. Besides that, the type of leather control the amount of enzyme needed (Varela et al. 1997; Rao et al. 1998)

2.2.3 Food industry

The major application of proteases in the dairy industry is in the manufacture of cheese. There are now three types of commercial milk-coagulating enzymes which are animal rennets, microbial milk coagulants, and genetically engineered chymosin. Both animal and microbial milk-coagulating proteases belong to a class of acid aspartate proteases. The development of microbial milk coagulants and chymosin are due to the shortage of rennets and the increased demand for cheese production. In cheese making, the primary function of proteases is to hydrolyze the specific peptide bond to generate para-k-casein and macropeptides(Rao et al., 1998).

Proteases are also used in the baking to modify gluten which present in wheat flour and has the ability to expand as bread dough rises during the baking process. The dough-making process can be accelerated by addition of proteases to partially hydrolyze the gluten. Typically, heat-labile fungal proteases are used for gluten hydrolysis in baking, such that the enzyme denatures as temperature rises in the early stages of baking(O.P.Ward et al., 2009).

Protein hydrolysates play an important role in blood pressure regulation and are used in infant food formulations, specific therapeutic dietary products, and the fortification of fruit juices and soft drinks. (Neklyudov et al. 2000).Many types of proteins, including soy protein, gelatin, caseins, and whey proteins may be modified using alkaline proteases. Microbial alkaline proteases and neutral proteases are commonly used for production of protein hydrolsates (O.P.Ward et al., 2009). Rebeca et al. (1991) reported the production of fish hydrolysates of high nutritional value, using B. subtilis proteases. Fujimaki et al. (1970) used alkaline protease for the production of soy protein hydrolysates. Perea et al. (1993) used alkaline protease for the production of whey protein hydrolysate, using cheese whey in an industrial whey bioconversion process.

2.3 Sources of protease

Since proteases are physiologically necessary for living organisms, they can be found in a wide diversity of sources such as plants, animals, and microorganisms. Plant kingdom occupies the topmost rank (43.85 %) for finding proteases, followed by bacteria (18.09 %), fungi (15.08 %), animals (11.15 %), algae (7.42 %) and viruses (4.41 %). Cysteine protease are produced abundantly (34.92 %) in plants whereas microbes have ability to secrete large quantities of serine (13.21 %) and aspartic (8.81%) proteases. Serine, cysteine and aspartic proteases are commonly present in animal. (Mahajan and Badgujar, 2010).

2.3.1 Production of Animal and Plant Protease

The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin, and rennins (Boyer 1971; Hoffman 1974). The production of animal proteases are depend on the availability of livestock for slaughter, which in turn is governed by political and agricultural policies (Rao et al., 1998). These animal proteases have wide application in industries.

The availability of land for cultivation, climatic conditions and time-consuming process are the factor that govern the use of plants as source of protease production(Rao et al., 1998). Plant proteolytic enzymes, such as papain from Carica papaya latex and bromelain from the pineapple family Bromeliaceae, have been produced commercially. Papain are widely used in the food industry (Neidlema 1991), beer clarification, (Caygill, 1979), meat tenderizing and preparation of protein hydrolysates (Dupaigne, 1973). Bromelain is derived from stem and juice of pineapple which is useful and effective in reducing inflammation associated with infection, sinusitis, osteoarthritis and cancer (Gautam et al., 2010).

2.3.2 Production of Bacterial Protease

Microorganism is an excellent source for protease production. Bacillus and Aspergillus species are attractive hosts for protease production due to their high growth rates. Bacillus spp. produces the alkaline serine proteases which are used extensively in detergents and in food processing whereas neutral proteases is useful in brewing and in general food processing. Fungal proteases are also produced commercially for food applications, with Aspergillus species as dominant producers. All of the predominant industrial microbial proteases are secreted as extracellular enzymes into the culture medium. (Rao et al., 1998).

2.4 Distribution of Protease producing bacteria

Bacteria were isolated and cultivated from all possible regions of the earth due to their wide range of habitat and diversity (Das et al., 2012). Zhou et.al (2009) reported that the richness of the cultivable protease-producing bacteria reached 106 cells/g in all sediments of the South China Sea. The predominant cultivated protease-producing bacteria were mainly affiliated with the class Gammaproteobacteria and grouped in the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera. Alteromonas (34.6%) and Pseudoalteromonas (28.2%) were the predominant groups. The other genera comprised between 2.6% and 7.7% of all strains. (Zhou et al., 2009).

Aftab et al (2006) reported that Bacillus sp. was found to be predominant in various soil fields of Karachi. 2% were found be B.coagulans, B.stearothermophilus and B. licheniformis, 4% were B. cereus and B.circulans, 6% were B.alvei, 8% were B.laterosporus, 10% were B. pumilus, 20% were B.brevis and 21% were B.sphaericus and B. macerans. Only 2% were found to be M.luteus(Aftab et al., 2006)

Halophilic protease producing Halobacterium spp were isolated from fermented fish samples (Kanlayakrit and Bovornreungroj, 2005). Halophilic protease producing Bacillus spp were found in sediment and water samples from different parts of Moharloo salt lake in Iran by Ghasemi (2011) (Ghasemi et al., 2011).

Alkaliphilic protease producing bacteria Bacillus halodurans, exhibit maximum activity at pH 10 with temperature 70 ??C, was isolated from water and soil samples collected from an Egyptian soda lake(Ibrahim et al., 2007). In addition, alkaliphilic Bacillus sp. with optimal activity at 50??C with optimum pH at 11.5 was found in soil samples from different habitats including, tanneries, soap factory, garden soil, soil composite and waste dumping areas in Pakistan (Khan et al., 2011).

Tang et al (2008) isolated solvent-tolerant Pseudomonas aeruginosa from crude oil contaminated soil samples located in Jiangsu province, China (Tang et al., 2008).

2.5 Identification of bacteria

Various phenotypic and genotypic methodologies are being used to identify and characterize unknown bacteria (Habib et al., 2012, Fetyan and Monsour, 2012). Phenotypic methods play a important role in identification but the molecular technique are more reliable and effective for identification and investigation of genetic diversity of bacterial isolates(Pace, 1997, Fetyan and Monsour, 2012). Tang et al.(1998) compared various identification systems such as cellular fatty acid profiles, carbon source utilization, and conventional biochemical identification with the 16S rRNA gene sequence to identify unusual aerobic gram-negative bacilli and coryneform organisms isolated from clinical specimens. He reported that16S rRNA gene sequence provided more rapid and reliable result than conventional methods.

2.5.1 Molecular technique

Woese et al. (1985) developed a new standard for identifying bacteria by comparing a stable part of the genetic code. Candidates for this genetic area in bacteria included the genes that code for the 5S, the 16S and the 23S rRNA.

Elsayed and Elbestawy (2008) stated that some unique part of DNA or RNA can provide the information on identity of organisms (Elsayed and Elbestawy, 2008).

Sequence analysis of the 16S ribosomal RNA (rRNA) gene has been widely used to identify bacterial species (Ibrahim et al., 2007, Brindha and Mathew, 2012, Das et al., 2012). Bacterial 16S rRNA genes generally consist of conserved region and hypervariable region. Conserved region is useful in studying the relationships among distant taxa among bacteria and enabling PCR amplification of target sequences using universal primers (Goebel et al, 1987). Hypervariable regions are flanked by conserved stretches in most bacteria which can be use in differentiation of species (Van de Peer et al.1996).

Sufficient size of 16s RNA (ca. 1500 bases) makes it easily to be sequenced. At the same time, this gene contain sufficient information in identification and phylogenetic analysis(Spratt, 2004).

The most direct and rapid way to identify the bacteria is to carry out PCR amplification of the 16sRNA by using primers from highly conserved flanking sequences (Saiki et al., 1988; Jensen 1993). PCR is a technique, which uses a DNA polymerase enzyme to make a huge number of copies of given piece of DNA or gene(Spratt, 2004).

The accurate 16S rRNA gene sequence identification of organisms is depend upon accurate sequences in databases, appropriate names associated with those sequences, and an accurate sequence for the isolate to be identified.(Clarridge, 2004).

2.5.2 Phenotypic techniques

Database with an accurate morphologic and biochemical description of typical strains and standard methods to determine these characteristics for the isolate to be identified are the requirements in order to identify the microorganisms by using phenotypic technique (Clarridge, 2004). Phenotypic techniques including Gram stain results, colony morphologies, growth requirements and biochemical reaction of microorganisms.

Some characteristics of microbial are no constant and can change due to stress or evolution; common microorganisms present with uncommon phenotypes; unusual microorganisms are not present in reference databases; databases are out of date are the drawback of phenotypic methods (Ochman at al., 2005; Petti et al., 2005)

Before start to examine the unknown bacterial isolate, the purity of the culture must be confirmed. The Gram-staining as the initial stage to observe the size, shape, and arrangement of cells (Holding and Collee, 1971). When undertaking the biochemical test, size of the inoculum, the volume of the test medium, or the type of container to be used are not under consideration. However, Holding and Collen (1971) stated that standard inoculum pipetted from a homogeneous suspension of the test organism are more reliable than a loopful of microbe from an agar slope or plate culture (Holding and Collee, 1971). Besides that, the stage of growth of the test inoculum may influence the result.

In some biochemical test such as voges proskauer test and methyl red test, the development of acidic or alkaline product by the microbes are indicated by the colour change of pH indicator incorporated in the test medium. Holding and Collee (1971) stated that indicator solutions should not be incorporated in strongly coloured culture media or in cultures that may develop colour as a result of bacterial pigment production because it will mislead the results. (Holding and Collee, 1971)

CHAPTER 3

MATERIAL & METHODS

3.1 General Methodology

3.1.1 Preparation of Media

The instruction was followed based on the label on the container of the medium. Dehydrate powdered-form media were weighed and dissolved in sterile deionised water. All of the media, agar and reagent were sterilized before using. Additional heat stable supplements were mixed well before autoclaved whereas heat labile compound were filtered and added to autoclaved media. Molten agar was poured onto petri dishes after autoclaving and allowed to solidify before being stored at room temperature. Methods for media preparation are stated in Table 3.1

Table 3.1 Preparation of Media Used in this Study

Media

Suppliers

Method of preparation

Gelatin medium

Gene Chem

13g of nutrient broth powder and 60g of gelatin powder was dissolved and topped up to 1L using sterile deionised water

Luria-Bertani (LB) agar

MERCK

37.0 g of LB agar powder was dissolved and topped up to 1L using sterile deionised water.

Luria-Bertani (LB) broth

MERCK

20g of LB broth powder was dissolved and

topped up to 1L using sterile deionised water.

Methyl Red- Voges Proskauer (MR-VP) broth

BD

17g of MR-VP broth powder was dissolved and topped up to 1L using sterile deionised water

Muller Hinton (MH) agar

Oxoid

38.0 g of MH agar powder was dissolved and topped up to 1L using sterile deionised water

O-F (Oxidation-Fermentation)

Basal Medium

BD

9.4g of OF powder was dissolved and topped up to 1L using sterile deionised water.

Phosphate buffered saline (PBS)

MP Biomedicals

One PBS tablet was dissolved in 100ml of

sterile deionised water

SIM agar

MERCK

30g of SIM powder was dissolved and topped up to 1L using sterile deionised water

Simmon???s Citrate agar

MERCK

24g of Simmon???s citrate agar powder was dissolved and topped up to 1L using sterile deionised water.

Skim milk agar

BD

30g of Difco Skim Milk Powder and 20g of agar-agar powder were dissolved and topped up to 500mL using sterile deionised water respectively

Starch agar

Sisco Research Lab Pvt Ltd

25g of starch agar powder was dissolved and topped up to 1L using sterile deionised water

Tributyrin agar

R&M Chemicals

23g of tributyrin agar base powder was mixed with10ml of tributyrin and topped up to 990mL using sterile deionised water

??Triple sugar iron (TSI) agar

MERCK

65g of TSI agar powder was dissolved and

topped up to 1L using sterile deionised water

Tryptone??broth

Oxoid

10.0g of tryptone powder and 5.0g of sodium chloride were dissolved and

topped up to 1L using sterile deionised water

Urea broth

MERCK

38.5g of urea broth powder was dissolved and topped up to 1L using sterile deionised water

3.1.2 Sterilization

Sterilisation of all media and reagents were accomplished either through

autoclaving or filter sterilisation. Heat stable media and reagent were autoclaved at 121??C at 15 psi for 15 minutes whereas heat-labile media or reagent such as urea broth and glucose were filter-sterilised using a 0.20 ??m syringe filter. Glasswares, pipette tips, microcentrifuge tubes and other apparatuses were autoclaved before using it.

3.2 Sample Collection

The water and soil samples were collected from different sites of University Tunku Abdul Rahman and Lata Kinjang. 100g of soil were collected approximately 0 to 6 cm below the surface and placed into a sterile plastic bag.

3.3 Isolation and Screening of Protease Producing Microorganisms

Protease producing bacteria were isolated from soil and lake water. Spread plate method was used to isolate the bacteria. Firstly, the dilution was carried out by adding 1 g of soil sample and 1ml of water sample into 9 ml of 1M of PBS, separately. They were then incubated overnight on shaker machine at 37C, 88rpm. Overnight incubated cultures were then subjected to a 100-fold serial dilution using PBS. A volume of 100 ??l of each dilution was poured and spread evenly on the surface of skim milk agar plate by using a sterile glass spreader. After the above procedures were completed, the plates were incubated in an inverted position at 37??C for 24 hours.

Protease producers among the microbes were identified by a clear zone of skim milk hydrolysis around the colonies. Isolates exhibiting the largest cleared zone around their colonies are picked up. One single colony was identified by gram stain and re-streaked as a primary inoculant on the surface of LB agar plate. The plates were then incubated at 37??C. Gram stain was undertaken to re-check the identical cell morphology and gram reaction comparing to the original colony.

3.4 Preliminary Characterization of Bacteria Isolates

The isolated bacteria were identified based on cellular morphology, gram staining and biochemical tests. Molecular identification was achieved by 16s rDNA sequencing will be carried out to further confirm the species of isolated bacteria.

3.4.1Colony Characteristics

Isolated bacteria were streaked on LB agar and incubated overnight. The colony characteristics of bacteria such as shape, elevation, edge, opacity, texture and color were observed.

3.4.2 Differential Staining ??? Gram???s Staining

The single colony of pure isolated bacterium was placed on the microscope slide by using inoculation loop. The bacterial smear was heat-fixed by passing the glass slide through the flame of the bunsen burner. The slide was allowed to cool and flooded with crystal violet for 60 seconds. The slide was then rinsed gently under running water by keeping the slide in tilt position at an angle of 45?? to remove the dye. It was then flooded with Gram???s iodine for 60 seconds and washed under running water. The stain was then decolorized with 95% (v/v) alcohol which was washed off immediately. Counterstaining is done with safranin for 1 minute and rinsed with water. The slide was then dried by using blotting paper and observed under oil immersion lens microscope. Colour and morphology of the bacterial cells were recorded.

3.4.3 Biochemical Test

3.4.3.1 Indole Production

The tryptone broth was prepared , sterilized and poured into tubes. The bacterial isolate was inoculated into tryptone broth and incubated at 37??C for 48 hours. Following incubation few drops of Kovac???s reagent were added. The test tube was observed for color reaction. Formation of a red or pink color ring at the top was taken as positive.

3.4.3.2 Methyl Red (MR) Test

The glucose phosphate broth was prepared, sterilized and poured into tubes. The bacterial isolate was inoculated in glucose phosphate broth and incubated at 37??C for 48 hours. Five drops of alcoholic methyl red solution were added to the culture, mixed well and the results were read immediately. Development of red color is taken as positive; yellow color indicates a negative test.

3.4.3.3 Voges-Proskauer Test

The glucose phosphate broth was prepared, sterilized and poured into tubes. The bacterial isolate was inoculated into glucose phosphate broth and incubated at 37??C for 48 hours. Then 0.6 ml of Barrit???s solution A (alpha-naphthol) and 0.2ml of Barrit???s solution B (40% KOH were added to the test broth and shaken to aerate the sample. The tube was allowed to stand for 15 minutes. A positive reaction was indicated by the development of a pink color or red colour.

3.4.3.4 Citrate Utilization Test

The Simmon???s medium was prepared, sterilized and poured into the tubes. The medium was allowed to cool and solidify in the form of slants with deep butt. The surface of citrate agar slope was inoculated heavily with the bacterial isolate by using inoculation loop and incubated at 37??C for 48 hours. The change of colour from green to blue indicated the positive reaction.

3.4.3.5 Catalase Test

The pure bacterial isolates were first streaked onto LB agar and allowed to grow overnight. The single colony of pure isolated bacterium was placed on the microscope slide by using sterile inoculation loop. A few drops of 3 % hydrogen peroxide (H2O2) were added to the colonies on the plate. Presence of bubbles indicated positive reaction.

3.4.3.6 O-F (Oxidation-Fermentation) Test

The OF basal medium supplemented with 1 % glucose was prepared, sterilized and poured into the tubes. Each bacterial isolate was then deeply stabbed into 2 tubes of O-F basal medium by using a needle. Sterile mineral oil (2-3ml) was added into one of the inoculated tube. The inoculated tubes were incubated at 37??C for 48 hours. A color change in medium, from green to yellow in both tubes after incubation indicated that the bacterium is a fermentative bacterium.

3.4.3.7 Gelatin Test

Gelatin medium was dispensed into test tube and sterilized. Each bacterial isolate was inoculated into tube of gelatin medium by using inoculation loop and incubated at 37??C for 5-7 days. Gelatin hydrolysis was indicated by liquefying of the medium after the tube was kept at 4??C for 30-40 minutes.

3.4.3.8 Motility and Hydrogen Sulfide (H2S) Test

The Sulfide Indole Motility (SIM) medium was prepared, poured into the tubes and sterilized. The medium was allowed to cool and solidify. Each bacterial isolate was then inoculated by stabbing a straight inoculation needle down the center of the tube of SIM medium and incubated at 37??C for 24 hours. The growth of bacterial isolate radiating out from the central stab line indicated that bacterium is a motile bacterium. For H2S test, a black deposit was considered as a positive reaction.

3.4.3.9 Urea Test

The Christensen's Urea Agar was prepared, sterilized and dispensed into the tubes. The medium was allowed to cool and solidify in the form of slant with deep butt. The surface of a Urea Agar slope was inoculated heavily with a pure bacterial isolate culture by using inoculation loop and incubated at 37??C for 48 hours. The positive result of urea test was indicated by observing the change of medium from yellow to pink color.

3.4.3.10 Starch Hydrolysis Test

The starch agar was prepared, sterilized and poured in a thick layer into sterile Petri disks. A single starch agar plate was divided into four quadrants for four different inoculations. Each hole in each quadrant was cut by using sterile cork borer. A fresh pure bacterial suspension was inoculated in hole of starch agar and incubated at 37??C for 48 hours. After that, the surface of starch agar was flooded with Gram???s iodine solution. The positive result was indicated by observing the clear zone formation around the inoculated holes.

3.4.3.11 Tributyrin Test

The tributyrin agar was prepared, sterilized and poured in a thick layer into sterile Petri disks. Each tributyrin agar plate was divided into four quadrants for four different inoculations. Each hole in each quadrant was cut by using sterile cork borer. A fresh pure bacterial susupension inoculated in hole and incubated at 37??C for 48 hours. The positive result was indicated by observing the clear zone formation around the inoculated holes.

3.4.3.12 Triple Sugar Iron (TSI) Test

The TSI medium was prepared, sterilized and poured into tubes. The medium was allowed to cool and solidify in the form of slant with deep butt. The surface of a TSI agar slope was inoculated heavily with pure bacterial suspension by using inoculation loop and incubated at 37??C for 24 hours. A change of color from red to yellow was considered as positive reaction.

3.5Physiological Characterization of Bacterial Isolates

3.5.1 pH

Three sets of skim milk agar plates with different pH (5, 7 and 9) were prepared accordingly. Each plate was divided into four quadrants for four different inoculations. Each hole was cut in each quadrant and inoculated with respective fresh bacterial suspension. Then, the plates were incubated at 37??C for 24 hours. After incubation, the plates were observed and the diameters of clear zone were then recorded accordingly.

3.5.2 Temperature

Skim milk agar plates were prepared. Each plate was divided into four quadrants for four different inoculations. Each hole was cut in each quadrant and inoculated with respective fresh bacterial suspension. Then, each of the plate with respective bacterial isolates was placed at each of the following temperature: 20??C, 30??C, 40??C and 50??C for 24 hours. After incubation, all the plates were observed and the diameters of clear zone were then recorded accordingly.

3.5.3 Carbon Sources

Four set skim milk agar supplemented with different 1% (w/V) carbon source which were glucose, fructose, sucrose and lactose were prepared and poured into sterile Petri disks. Each plate was divided into four quadrants for four different inoculations. Each hole was cut in each quadrant and inoculated with respective fresh bacterial suspension. Then, the plates were incubated at 37??C for 24 hours. After incubation, the plates were observed and the diameters of clear zone were then recorded accordingly.

3.6 Antibiotic Susceptible Test

0.85% of NaCl solution were prepared and poured evenly into each tube. The bacterial isolates were inoculated in LB agar and incubated at 37??C overnight. After incubation, the colonies of respective bacterial isolate were transferred into tube of 0.85% of NaCl solution. McFarland standards was used as turbidity standards for the preparation of suspensions of microorganisms. The density of these suspensions was adjusted to 0.5 McFarland standards. A sterile cotton swab was dipped into the inoculums was then swabbed 3 times over the entire Mueller- Hinton (MH) agar surface by rotating the plate approximately 60?? each time to ensure an even distribution and confluent growth. Four different commercial antibiotic discs were used in this study which were ampicillin, tetracycline, chloramphenicol and doxycycline. The discs were impregnated on the inoculated agar approximately 2 cm from the edge of the plates by using a sterile forceps. After that, the plates were incubated overnight at 37??C. The All the plates were observed for the presence of inhibition zone around the discs and recorded.

3.7 Molecular Identification

Sequencing of 16S rDNA was used for genotypic characterization. There were four major steps, including genomic DNA extraction, Polymerase Chain Reaction (PCR) amplification of 16S rRNA, sequencing of PCR amplicon, and 16S rRNA sequence analysis.

3.7.1 Genomic DNA extraction

Genomic DNA of the selected isolate of protease producing bacteria was extracted using fast boil method. The respective bacterial isolate was cultured overnight in 1ml LB broth at 37 ??C with 100rpm agitation. After incubation, 500??l of bacterial culture were transferred into new micro centrifuge tube and centrifuged at 14,000 rpm for 2 minutes at room temperature. The supernatant were discarded whereas the pellet was then re-suspended with 50??l of sterile distilled water. The suspension was then heat at 70??C for 30 minutes. After that, the sample was centrifuged at 14,000 rpm for 2 minutes. The supernatant was then transferred to a new sterile micro-centrifuge tube and stored at -20??C until future use. The concentration and purify of DNA obtained were measured using Nanodrop 1000 (Thermo Scientific).

3.7.2 Polymerase Chain Reaction (PCR)

3.7.2.1 Primers for PCR Amplification

Amplification of the 16S rDNA bacterial genes was carried out using the universal bacterial primers. Sequences of the primers were stated in Table 3.2

Table 3.2Primers Sequences used in16s rDNA Gene Amplification

Primer

Primer Sequences (5??? to 3???)

Length (base)

338F

5'- ACTCCTACGGGNGGCNGCA-3'

19

515R

5'- GTATTACCGCNNCTGCTGGCAC-3'

22

Legend : F: Forward; R: Reverse

Note : N:A/G/C/T

3.7.2.2 Preparation of PCR mixture

Each 25 ??l of reaction mixture contained 2 ??l of genomic DNA, 18 ??l of dH2O, 2.5 ??l of 10x buffer, 0.5??l of dNTPs mixture (10 mM each concentration), 0.5 ??l of each forwar and reverse primer and 1 ??l of Taq DNA polymerase.

3.7.2.3 Condition of PCR Amplification

Amplification of 16s rDNA was performed on a Thermal Cycler (Bio-Rad) using the conditions state below:

94??C : 5 minutes Initial denaturation 1cycle 94??C : 45 seconds

60??C : 45 seconds Annealing temperature 30 cycles

72??C : 45 seconds

72??C : 10 minutes Final extension 1 cycle

3.7.2.4 Gel electrophoresis of PCR Products

An aliquot of 1 ??l of the PCR product was mixed with 1??l of loading dye and 4??l of dH2O. The samples were then loaded into separate wells in 2% (w/v) agarose gel and analyzed using electrophoresis. The size of PCR products was compared with 100bp DNA ladder. The gel was stained by ethidium bromide and the PCR product was then visualized under the UV transilluminator. The expected size of the PCR products was approximately 200 bp.

3.7.3 Purification of DNA

PCR products to be used for sequencing were purified using Gel/PCR DNA fragment extraction kit (Geneaid). 5 volumes of DF Buffer were added to 1 volume of PCR product and mixed gently by inverting the tube several times. The DF column was inserted into a 2 ml collection tube. The sample was then transferred to DF column and centrifuged at 15000 x g for 35 seconds. The flow-through was discarded and the column was placed into the same collection tube. 600??l of Wash buffer were added into center of DF column and centrifuged at 15000 x g for 35 seconds. The flow-through was discarded and the column was placed into the same collection tube. An additional centrifuge at 15,000 x g rpm for 3 minutes was performed to remove the residual ethanol completely. The column was placed on a sterile 1.5 ml micro-centrifuge tube. An amount of 35 ??l of Elution Buffer was added into the center of the column matrix, allowed to stand for 10 minutes and centrifuging at 15,000 x g for 2 minutes. Eluted DNA was stored at -20??C.

3.7.4 DNA Sequencing and Analysis

3.7.4.1Sequencing of the purified 16s DNA

Extracted 16sDNA was outsourced to Medigene?? Sdn. Bhd. for sequencing using specific primers, 338F and 515R.

3.7.4.2 Blast Analysis

By receiving the results, the 16S rDNA nucleotide sequence of isolate has been deposited in GenBank and aligned with the 16S rRNA sequences available in nucleotide database in NCBI, (National Center for Biotechnology Information, Available at:http://www.ncbi.nlm.nih.gov/), using BLAST software, (Basic Local Alignment Search Tool)