Investigating extracellular Enzymes of microorganisms

Introduction to Microbial Enzymes Microbial enzymes have applications in many fields  Organic synthesis  Clinical analysis  Pharmaceuticals  Detergents  Food production  Fermentation Hwa Chong Institution Projects' Competition 2

Introduction to Microbial Enzymes 3 Hwa Chong Institution Projects' Competition Extracellular microbial lipase  Catalysts for interesterification of oils and fats (Macrae, 1983) for use in synthesis of biodiesel  Lipases from Pseudomonas have potential in biotechnology (Gilbert et al., 1991) Microbial proteases  Hydrolysis of proteins  One of the most important groups of industrial enzymes (Wu, 2006)

Rationale Problems with currently available microbial enzymes.  Most are from fungus (Aspergillus niger) or bacterium (Bacillus subtilis)  Problem with biosafety of these organisms Hwa Chong Institution Projects' Competition 4

Objectives 5 Hwa Chong Institution Projects' Competition

Hypothesis A few species of bacteria produce extracellular protease and lipase that are secreted into the culture supernatant. The protease and lipase have specific optimal pH Hwa Chong Institution Projects' Competition 6

Variables Independent variables Test organisms  Escherichia coli MM294  Micrococcus luteus ATCC4698  Staphylococcus epidermidis ATCC12228  Serratia marcescens D1 Medium used for detection of enzymes Protease  LB + gelatin, LB + bovine serum albumin Lipase  LB + olive oil, LB + palm olein

Variables Dependent  Presence or absence of clear zone around well  Diameter of clear zone created Hwa Chong Institution Projects' Competition 8 Controlled  Volume of culture supernatant filled into wells  Temperature of bacterial growth

Materials and apparatus 9 Hwa Chong Institution Projects' Competition Materials Escherichia coli MM294 Micrococcus luteus ATCC4698 Staphylococcus epidermidis ATCC Serratia marcesens D1 LB broth and agar, agar powder Gelatin, bovine serum albumin Olive oil, palm olein 2% sodium alginate 0.1 M calcium chloride, 0.85% sodium chloride solutions Sterile toothpicks Apparatus Shaking incubator Incubator Centrifuge Sterile Forceps Micropipette

Method - Preparation of agar 10 Hwa Chong Institution Projects' Competition Agar for testing of protease  LB + 1% Gelatin  LB + 1% BSA  Plain Agar + 1% Gelatin  Plain Agar + 1% BSA Agar for testing of lipase  LB + 1% Olive Oil  Plain Agar + 1% Olive Oil  LB + 1% Palm Oil  Plain Agar + 1% Palm oil

Method – Spot inoculation of bacterial cells Spot Inoculate bacteria on LB + Gelatin / LB + BSA Incubate plates at 37 degrees celsius Stain agar plate with Coomassie Blue and observe for clearing zones 12 Hwa Chong Institution Projects' Competition

Inoculate bacteria into 5ml LB broth and incubate at 37 degrees Celsius with shaking Centrifuge at 5000rpm for 10 minutes Punch wells in agar plates (Plain Agar + 1% Gelatin/ 1% BSA) Fill wells with 70µl Culture supernatant and 70µl LB Broth as control Incubate plates at 37°C Observe for zone of clearing after staining with Coomassie Blue Hwa Chong Institution Projects' Competition 13 Method – Well diffusion test

Method - Serial dilution test The bacteria was inoculated into LB broth and grown at 30°C Next, it is taken out the next day and its absorbance was recorded. Absorbance was taken at 600nm Serial 2-fold dilution on culture supernatant until 1:256 70µl diluted supernatant (test) & 70µl LB broth (control) were filled into wells Hwa Chong Institution Projects' Competition 14

Method – Effect of pH on protease activity S. marcescens was grown overnight in 10 ml LB broth Culture supernatant was obtained by centrifuging at 7000 rpm for 10 min pH of culture supernatant was adjusted with acid or alkali to pH 3, 5, 9,  L of supernatants of varying pH were filled into wells of gelatin agar plates and incubated at 30  C

Methods – Immobilisation of culture supernatant Equal volumes of culture supernatant of (test) or LB broth (control) and sodium alginate were mixed well Mixture was released dropwise into calcium chloride solution Beads formed were rinsed with sodium chloride solution Test beads Control beads

Method – Hydrolysis of gelatin by immobilised cells 10 ml of 1% gelatin is pipetted into 6 sterile Falcon tubes.Place 50 beads containing supernatant each into a tube (x3 tubes).Place 50 beads containing LB broth (control) each into a tube (x3 tubes). Incubate tubes at 30  C with shaking overnight. Bradford test was carried out to measure total proteins as follows. Remove 100  l of 1% gelatin and add to 1.9 ml of Bradford reagent. Read absorbance at 595 nm. Blank- 100  l of water ml of Bradford reagent. Hwa Chong Institution Projects' Competition 17

References Gisela Kouker &Karl-Erich Jaeger (1987). Specific and Sensitive Plate Assay for Bacterial Lipases. Applied and Environmental Microbiology, 53, Retrieved February 12, 2010 from Yoshitaka Kokusho, Haruo Machida and Shinjiro Iwasaki (1982). Studies on Alkaline Lipase: Isolation and Identification of Lipase Producing Microorganisms. Agric. Biol. Chem.,46, Retrieved February 12, 2010 from =1159&lang=en&from=jnlabstract =1159&lang=en&from=jnlabstract E. Jane Gilbert, Jane W. Drozd and Colin W. Jones (1991). Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2. Journal of General Microbiology, 137, Retrieved February 12, 2010 from Macrae A.R. (1983). Lipase-Catalyzed Interesterification of Oils and Fats Journal of the American Oil Chemists' Society, 60, Retrieved February 10, 2010 from Hasan Fariha, Aamer Ali Shah and Abdul Hameed (2005). Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, Issue 2, Retrieved February 10, 2010 from 1&_user=10&_coverDate=06%2F26%2F2006&_rdoc=1&_fmt=full&_orig=search&_cdi=5241&_sort =d&_docanchor=&view=c&_searchStrId= &_rerunOrigin=scholar.google&_acct=C &_version=1&_urlVersion=0&_userid=10&md5=49855f7cf38c5b0bf920d9bc491b &_user=10&_coverDate=06%2F26%2F2006&_rdoc=1&_fmt=full&_orig=search&_cdi=5241&_sort =d&_docanchor=&view=c&_searchStrId= &_rerunOrigin=scholar.google&_acct=C &_version=1&_urlVersion=0&_userid=10&md5=49855f7cf38c5b0bf920d9bc491b Hwa Chong Institution Projects' Competition

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