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A STUDY Oli' CERTAL~ Fi:.CTGHS ii.l?FF:CTiilG 'l'EE hWDUCTICH Ai-JD ACTIVIT{ vr' BLG'l'EHIAL A:. :YLASE:J P:WDUCFJJ BY ORGANIS!'1S GROHIT rn A SYNTHETIC 1.1:DIA By : ,::~l a,, Georc e Sch;,d.dt /1 Dr,chel or of Sc:1..cnce Stillwe.t er, 01:l aho:·La 1951 ~ub~1itted t o the fnc i_; J.t t of the Gntc1 uc.te Sch~ol of the Oklahoma AgricuH,1u·a l and rfocha :,1:~c ·.::i.. College j_n pEtrtia l f ,.11 fill>1ent of the requirel'lents for t. he (Je e;ree of l-1ia::iT~i l. O•' ·jGIErJCE j-<1: , 1953 Ml ~MO~l AGP.ICULTURAl & l'lf.C~t.NIC&l C8LLEGE Ll E R. ARY DEC 10 1953 A STUDY OF CERTAIN l<"'ACTORS AFFECTING THE PRODUCTION AND ACTIVITY OF BACTERIAL AMYLASES PRODUCED BY ORGANISMS GR&N IN A SYNrHETIC MEDIA Thesis Approved: Dean of the Graduate School ii 30 905B ACICNOWLEIXtEMENTS I would like to express my sincere appreciation to Dr.T.L.Johnson for his assistanGe and wise counsel during the oourae of this work.and also to Mias Zana Skidmore,.Dr.E.E.Harndan,and Mr.Robert D. Conrad. all or whom provided valuable advioe and willing assista..-ioe.I am con siderably indebted to Mr. Willard E. Hardwiok,who first undertook in vestigations of this nature at this institution. I would like to t hank very particularly my father,whose combination or sound advice,finanoial aid,and perseverance enabled me to complete this degree. Last but by no means lea.st.I would like to t hank my wife.It was t:hrough her encouragement t }-i..at I became resolved to complete this t hesis and Degree. iii TABLE OF CONTENTS Chapter Page LIST OF TABLES. V I INTRODUCTION. 1 II LITERATURE REVIEW 2 III EXPERIMENT AL . 5 A. Normal Values of Growth and Amylase Production 5 1. Measurement of Bacterial Numbers. • 5 2. Colorimetric Determination of Starch Utilization. • . • • • • . 5 J. Normal Values of Growth and Amylase Production • • . • • • • • . 9 B. Effects of Salts in Low Concentration. • 11 1. Procedure . • . • . • • 11 2. Results . • . • . • • . • 12 C. Optimum pH and Optimum Temperature 15 1. Optimum pH. • . • . 15 2. Optimum Temperature 16 IV DISCUSSION 18 V SUMMARY 22 VI BIBLIOGRAPHY. 24 VITA .... 29 TYPIST PAGE 30 iv • LIST OF TABLES Table Page I Graph to Show that Beer's Law is Applicable to Starch Iodine Color Reaction . , , • . • • • • • • • II Growth of Organisms in Synthetic Media as Determined by Plate Count . • . 10 III Reduction of Starch in Inorganic Media 10 IV pH of Culture Fluid at Time That all Starch had Been Reduced . • . • . • . o • • • • 11 V Bacterial Numbers in Media Containing 0.25% NaCl 13 VI Amount of Starch Remaining in Culture Flask. Media C0ntaining 0.25%. • 13 VII Bacterial Numbers in Media Containing 0.1% KCl VIII Amount of Starch Remaining in Culture Flasks. Media Containing 0.1% KCl •...•..•.. 14 IX Time in Minutes Required for 50 ml. of Enzyme Filtrate to Reduce 0.001 gm. of Starch at various ph levels . o • 17 X Time Required for 50 ml. of Enzyme Filtrate to Reduce 0.001 gm of Starch at Various Temperatures (in M·inute s) . Cl • • • • • • • • 17 V I INTRODUGTICi'l' The purpose of this investigation was to make certain qualitativa and quantitative studies of factors affecting the production and act i vity of .3acterial Amylases from 18 strains of orgEnisms isolated by Hardwick in 10. ,;·.'. Hardwick isolated and tentatively identified 18 cultures of micro- organisms which evidenced amylolytic activity when grown upon a medium containing starch as the only organic constituent. PrelL~inary tests using the media recommended by Hardwick1 indicated t hat t he turbidity of the media would interfere ~ith testing procedures. A simpler, less turbid media was devised.2 This media proved to be satisfactory in all respects. This work includes; (1) A study of the effect of various concentra- tlons of sodium chloride and potassium chloride upon the rate of growth of the organisms and their capacity to produce amylase; (2) Determination of the optimum pH and optimum temperature of amylolytic activity; (3) The quantitative measurement of amylolytic conversion of starch by the use of a modified photocolorimetric method. 10,.ielianski I s Broth, with starch substituted for cellulose. ·sodium A.rnmonium Phosphate 1. 5 gm. Monopotassium phosphate 1.0 gm. Magnesium Sulphate 0.2 gm. Faultless Starch 2.0 gm. Water to one liter 1 II LITERATURE REVIEW The literature of starch fermentations and starch chemistry dates to the beginnings of civilization. Brewing was one of the first sciences known to man. Records indicate that the preparation of malt was known as early as 7000 B. C. Beer brewing had become an established industry among the Phoenecians and Egyptians by 5000 B. C. These ancients knew nothing of the conver sion of starch to sugar by malt. They only knew that malt was necessary if they were to obtain the desired yield of alcohol. (Lazar, 1938). The earliest scientific research concerning amylolysis occurred in 1815 when Kirchoff isolated the active ingredient of barley malt. In . - - . - .. 1817, Clement asserted that the fermentation of starch without conversion to intermediate sugars was possible. In 1833, Payen and Persoz conducted classic research in concentrating the enzyme by precipitation with alcohol and following its activity through the use of the starch-Iodine color reaction. Marker (1878) theorized that there were two enzymes present in malt and from his work arose the realization of the existance of alpha- and beta-amylase. The microbiology of starch was first investigated by deSaussure who 'inl819 reported the seemingly spontaneous decomposition of a starch . .. paste which had been le.ft in the open. Debrunfaut (1847) proved that the sugar obtained from starch by the action of malt was not, as pre- iously believed, glucose, but was instead a new sugar which he named maltose. O'Sullivan (1872-1878) expanded this work and proved that maltose was a disaccharide with nearly twice the rotatory power of glucose. 2 3 Atkinson (1881) studied the enzyme active on rice starch used in the preparation of a rice ba~r. This enzyme he called Koj'i Diastase. Previous to 1890, a number of incidental notations on the fermenta tion of starch by micro-organisms had been made. The first organized investigation of the reduction of starch by micro-organisms was conducted in organisms toward starch. Takamine (1898) prepared an amylase of fun gal origin which he named Taka-diastase. Mu,enter (1910) studied certain characteristics of Taka-diastase in detail. Schardinger (1901-1911) cultivated Bacillus macerans on certain starch media and obtained from this organism an amylase which would reduce starch to crystallin or Schardinger dextrins. The Schardinger dextrins consist of two water soluble, non-reducing dextrins which can be easily detected. The two dextrins were named arbitrarilr alpha- and beta Schardinger dextrins. Commercial preparations of bacterial anzy-lases were first made in 1917 from Bacillus sugtilis by Boiden and Effront. In the period 1917- 1931, several detailed investigations upon the amylases of Bacillus subtilis and Bacillus mesentericus were made. Among the most notable of the recent investigators into the pro duction and properties of bacterial a.my-lases are Tarr (1934), . Tilden and Hudson (1939), Kerr (1942, 1943), Wilson, Schoch, and Hudson (1943), Balls and Schwimmer (1944), Peltier and Beckford (1945), Beckford, Kneen, and Lewis (1945), Schwimmer (1945), Beckford, Peltier, and Kneen (1946), and Kneen and Beckford (1946). The media, experimental methods, and micro-organisms used by these . ecent workers differ widely. The media used ranged from a potato slurry (Tilden and Hudson) to a wheat bran mash (Beckford, Kneen, and Lewis). The micro-organisms employed included Bacillus subtilis, 4 Asper gillus oryzae, Bacillus polym..yxa, Bacillus macer ans, .BacilJ,1,1£1 graveolens, Bacillus mesentericus, Bacillus rn.ycoides, and spores of Ba cillus subtilis. The methods of determining the properties of bacterial amylases vary with the purpose and whim of the investigator. There is no standardiza tion of methods or values. Each worker devises his own method or modifies another method to suit his own purposes. Of necessity, each investigator, when measuring a particular property must devise an arbitrary unit of measure. This consistant inconsistancy renders evaluation of previous work difficult. III EXPERIMENTAL A. Normal Values of Growth and Amylase Production Before an evaluation of the amylase production of these organisms could be made, it was first necessary to est;ablish standards or normal values of quantitative and qualitative nature and devise methods whereby both the normal values and experimental results could be measured. 1. Measurement of Bacterial Numbers. The plate count was used to determine bacterial numbers. 2. Colorimetric Determination of Starch Utilization. One of the prerequisites for successful colorimetry is the use of a fluid which is not too dense to allow light rays to pass through. The media used by Hardwick was so turbid that it was impossible to use this media as a part of a colorimetric determination. It was necessary, there fore, to devise a media which contained no organic constituents except starch, which was relatively clear in character, and which would support the growth of the organisms used. A media meeting these requirements is described in the introduction. For determining quantitatively the amount of starch remaining in the media, colorimetry was chosen because colorimetric methods are usually fast, relatively simple, and reasonably accurate. Colorimetry cannot be used if the color intensity and the concentration of the unknown are not propo~tional, or do not follow Beer's law. The transmittance of a solution c0 .<.t aining light absorbing particles is dependent upon (1) the nature of ::ae substance, (2) the wavelength of light, (3) the amount, or concentra tion, of light absorbing particles in the solution.

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