PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR PHYTASE FROM PSEUDOMONAS SP. WEERACHAT SRI-AKKHARIN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (BIOTECHNOLOGY) FACULTY OF GRADUATE STUDIES MAHIDOL UNIVERSITY 2004 ISBN 974-04-5062-8 COPYRIGHT OF MAHIDOL UNIVERSITY Fac. of Grad. Studies, Mahidol Univ. Thesis / iv PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR PHYTASE FROM PSEUDOMONAS SP. WEERACHAT SRI-AKKHARIN 4536525 SCBT/M M.Sc.(BIOTECHNOLOGY) THESIS ADVISORS : SAOVANEE DHARMSTHITI, Ph.D. (GENETICS), SITTIWAT LERTSIRI, Ph.D. (AGRICULTURAL SCIENCE), SUCHAT UDOMSOPAGIT, Ph.D. ( MICROBIOLOGY) Abstact A bacterial strain capable of producing an extracellular phytase was isolated from soil. According to its colony morphology, Gram stain, biochemical characteristics, and ability to produce greenish pigment and growth on a selective medium, it was identified as Pseudomonas aeruginosa strain K2. Maximum phytase activity was obtained when it was cultivated in a 250-ml Erlenmeyer flask containing 75 ml of LB (Luria Bertani ) medium with initial pH of 5 under 200 rpm agitation at 400C for 24 hours. General properties of crude K2 phytase produced in LB medium were determined. Crude K2 phytase exhibited maximum activity at pH 5.0 and 450C. The enzyme was stable at acidic pH range of 3-5 and at temperatures lower than 400C. A study on the effect of EDTA (a chelating agent) and metal ions showed that the enzyme was stable with EDTA, Ca2+, Fe2+ and Fe3+ but it was moderately sensitive to Cu2+ and was strongly inhibited by Zn2+. Addition of Fe2+ to the crude phytase helped increase its temperature stability. This enzyme was sensitive to trypsin and bile acids, i.e. taurocholic acid and deoxycholic acid, whereas it was highly stable with lactic and propionic acid at either 0.1%(v/v) or 1.0%(v/v) concentration. Adding lactic and propionic acid could also promote the enzyme activity. K2 phytase could hydrolyse a range of non-phytate-based phosphorylated substrates, but only at lower activities than phytate. The crude enzyme also showed protease, alkaline and acid phosphatase activities at level of 4.10, 0.43 and 1.29 U/ml, respectively, and some carbohydratehydrolyzing enzymes at low levels of activity. Insensitivity to 2-mercaptoethanol indicated the absence of essential disulfide bond(s) in this phytase molecule. A complex medium, WTY, suitable for K2 phytase production has been developed. It was composed of compositions of PSM (Kerovuo et al., 1998) with reduction of glucose concentration to 1.5% (w/v) and omission of phytate in the formulae, and supplemented with 2.0% (w/v) yeast extract, 0.1% (v/v) Tween-80 and 10% (v/v) of wheat bran extract. Digestion of mixed chicken feed, rice bran, wheat bran and soybean meal by crude K2 phytase at 5 U per 1 g of such materials could markedly increase phosphate content and also increase reducing sugar and protein content. This digestion efficiency suggested that this enzyme was a potential candidate for use in animal feed applications. KEY WORDS : EXTRACELLULAR PHYTASE / Pseudomonas / PRODUCTION / CHARACTERIZATION 127 pp. ISBN 974-04-5062-8 Fac. of Grad. Studies, Mahidol Univ. Thesis / v Pseudomonas sp. (PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR PHYTASE FROM PSEUDOMONAS SP.) µ¦¨·Â¨³«¹¬µ»­¤´· °Á°Å¤rÅ¢Á­µÁºÊ° ª¸¦µ«¦¸°´ ¦· ¦r 4536525 SCBT/M . .( ª ¤ ÁÃ襸¸ª£µ¡ ) ³¦¦¤µ¦ª»¤ª·¥µ·¡r Ph.D. (Agricultural Science), : , Ph.D. (Genetics), Á­µª¸¥r ¦¦¤­·· , ­··ª´r Á¨·««·¦· , Ph.D. (Microbiology) ­»µ°»¤Ã­£· ´¥n° K2 Á}¸Á¦¸¥­µ¥¡´»r¸É­µ¤µ¦Â¥¤µÅoµ· ­µ¤µ¦¨·Á°Å¤rÅ¢Á­°°¤µ °Á¨¨rÅo µ¨ °µ¦¥o°¤­¸¼¨´¬³ °Á¨¨r µ¦­°µ¸ªÁ¤¸ ¨³ªµ¤­µ¤µ¦Ä µ¦­¦o µ ¦ª´ » ­¸ Á ¸ ¥ ªÂ¨³Á¦· °µ®µ¦· ¡· Á«¬­Î µ ®¦´ ÁºÊ ° Î µÂÁºÊ°ÅoÁ} ¤¨ ¹É¤¸¦³´ °»®£¼¤· 0 Pseudomoas aeruginosa pH Ä Pseudomonas ¨³¡ªnµµ¦Á¡µ³Á¨¸Ê¥ÁºÊ°Ä°µ®µ¦ Î µ Ä®o ­ µ¤µ¦ LB ¦·¤µ¦ Erlenmeyer flask µ ¤¨ ¸ÉÁ ¥nµoª¥ªµ¤Á¦Èª ¦°µ¸ ¸É C Á} Áª¨µ ¤ ¡ªnµÁ}­£µª³¸ÉÁ®¤µ³­¤Äµ¦Á¦·Â¨³­¦oµÅ¢Á­ µµ¦ «¹ ¬ µ » ­ ¤ ´ · ° Á°Å ¤ r ¹É ¥´ Å ¤ n n µ µ¦Î µ Ä®o ¦· ­» ·Í ¡ ªn µ Á°Å¤r ¤¸ ¦³­· · £ µ¡Äµ¦ Î µµ­¼­»¸É pH ¨³¸É°»®£¼¤· Ħ·¤µ ¨³Ä­£µª³¸ÉÁ}¦ Ã¥Á¡µ³¸É ¨³ Á°Å¤rÁnÁ¸¥ª´ Fe3+ Ä ³¸É trypsin, taurocholate % (v/v) ¨³¥´¡ªnµÁ°Å¤r­µ¤µ¦¦´¬µ·¦¦¤ÅªoÅoÄ ¸ C nª°»®£¼¤·ÎÉ µªnµ EDTA, Ca2+, Fe2+ C Zn2+ ¨³ ¨³ Cu2+ pH Á°Å¤r¸Ê¤¸ªµ¤n° ¤¸ ¨¥´¥´Ê°¥nµ´Án°·¦¦¤ ° deoxycholate µ¦Á·¤ lactic ®¦º° propionic acid ¨ÄÁ°Å¤r¡ªnµ­µ¤µ¦nª¥Á¡·É¤·¦¦¤Â¨³Á­¸¥¦£µ¡ °Á°Å¤rÅo oª¥ °µ¸Ê¥´¡ªnµÁ°Å¤rÅ¢Á­¸Ê­µ¤µ¦¥n°¥­µ¦¦³°¢°­Á¢·°ºÉÇÅo Ân¥n°¥Åo Ħ³´ ¸É ÎÉ µ ªn µ Á¤ºÉ ° Á¸ ¥ ´ µ¦¥n °¥Å¢Á æ¸ Á°­ ¢°­¢µÁ­ °µÅ¢Á­Â¨o ª ¥´ ¡Á°Å¤r · °ºÉ ÇÅo  n ¨³Á°Å¤r¨»n¤¸É­µ¤µ¦¥n°¥­µ¦¦³°Î µ¡ªµ¦rÃűÁ¦°¸oª¥ µ µ ¦ ¦´ ¦» ­¼ ¦°µ ®µ¦Î µ Ä®o Å o ° µ®µ¦¸É Á ®¤µ³­¤­Î µ ®¦´ µ¦¨· Å¢Á­µÂ¸ Á ¦¸ ¥ ­µ¥ ¡´ »r¸Ê ¹ÉÁ}°µ®µ¦ extract YY PSM ¸ÉŤnÁ·¤Å¢Á ¨³¨¨¼Ã­Á} ¨³Á·¤Á¡·É¤oª¥ Tween-80 ¨³ÎÊ µ­´µ¦Î µ oµª­µ¨¸ YY °µ¸Ê¥´¡°¸ªnµµ¦Ä­n Á°Å¤r ¨ ÅÁ¡ºÉ °¥n °¥Å¢ÁÄ°µ®µ¦Ån ¦Î µ o µ ªÁo µ ¦· ¤µ¢°­Á¢ÅoÁ}°¥nµ¸ ¦ª¤´Ê¥´­µ¤µ¦Á¡·É¤ 127 ®oµ yeast ISBN 974-04-5062-8 ¦Î µ o µ ª­µ¨¸ reducing sugar ¨³´É ª Á®¨º ° ­µ¤µ¦Á¡·É¤ ¨³SURWHLQFRQWHQW°¸ oª¥