Table Of ContentPURDUE UNIVERSITY
THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION
BY__________________EG-ON STARK___________________
entitled SJENQTHERMQPHILIG BACTERIA AND . THEIR
_________________ STARCH-SPLITTING ENZYMES______
COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES
AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS
FOR THE DEGREE OF
________________Doctor of Philosophy__________
rP. a.
Professor in Charge of Thesis
(P>^ .TetrauLI
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' / I V _ Heap of School or D epartm ent
(J.SJCarlifcg)
August 4--- 12-51-
TO THE LIBRARIAN;--
THIS THESIS IS^0T TO BE REGARDED AS CONFIDENTIAL.
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PBOFESBOB IS OBABOB
(P.A .Tetrault)
OBAD. SCHOOL FORM 8
STEN OTHERMOFHI LI C BACTERIA AND THEIR
STARCH-SPLITTING ENZYMES
A Thesis
Submitted to the Faculty
of
Purdue University
by
Egon Stark
in partial fulfillment of the
requirements for the Degree
of
Doctor of Philosophy
August 1951
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ABSTRACT
STEN OTHERMOFHI LIC BACTERIA AND THEIR STARCH-SPLITTING-
ENZYMES
Only three reports in the literature described in detail
the activity of stenothermophilie bacteria on starch. Cool-
haas (1928) studied the chemical changes brought about in
the medium. Imseneoki, Solntzewa and Kuzyurina (1942) and
Proskuryakov and Dimitrievskaya (1949) were the first ones
to isolate and test cell-free amylase preparations obtained
from the medium at 60 C.
A research project was undertaken to (a) provide a
collection of stenothermophilie, amylolytic ~bacteria,
(b) elucidate the conditions necessary for the formation of
amylase, and (c) isolate and characterize the amylolyti0
enzyme or enzymes.
(a) Thirty-four strains of stenothermophilic, amylolytic
bacteria, isolated from muck soil of Northern Indiana, were
studied determinatively by the methods of Gordon and Smith
(1949)• These cultures were almost identical with Bacillus
stearothermophilus ATCC#7954. The species pattern of
B. stearothermophilus as defined by Gordon and Smith was
modified to read:
Bacteria growing at 65 C, forming bulging spores,
hydrolyzing gelatin and starch, coagulating milk, forming
EgS in tryptone broth, growing on nutrient, glucose and
soybean agars, respectively, not growing on citrate, lead
acetate and stock culture agars when tested at 65 C, may
be considered as belonging to Bacillus stearothermophilus»
(b) All cultures hydrolyzed 0.5 per cent potato, soluble,
corn, rice and arrowroot starch in 1 per cent trypticase -
0.5 per cent yeast extract broth. The starches were attacked
regardless of the brand of the manufacturer and of the batch
used. However, results could not be duplicated with respect
to the ease of initial attack and with regard to the extent
of subsequent hydrolysis.
Strain differences were pronounced, because some cultures
hydrolyzed readily 10 per cent soluble starch, while others
attacked 0.5» but not 1 per cent. Length of the incubation
was important. Starch broth was a better medium than starch
agar. The initial pH of the medium influenced results.
Acid conditions (pH 6) favoured the initial breakdown of
starch, while pH 8 favoured the extent of subsequent hydro
lysis. Growth of most strains was inhibited at pH 5 and 9.
Changes of the reaction of the medium did not influence the
course of starch hydrolysis. Three cultures never turned
the medium acid, going from pH 7 to 8.4, yet, hydrolyzed
the starch.
The type of protein medium used affected the formation
of amylase very strongly. On 1.5 per cent trypticase 3 per
cent, on yeast extract 11 per cent, on protone 25 per cent,
on peptone 31 per cent and on tryptone 46 per cent of the
cultures failed to hydrolyze 1 per cent soluble starch. The
addition of 0.5 per cent yeast extract to 1 per cent of the
above protein media depressed the hydrolysis of starch in
trypticase (20 per cent negative), but enhanced it in the other
media: 17 per cent negative in protone, 23 per cent negative
in tryptone and 26 per cent negative in peptone broth.
Criteria selected to detect the hydrolysis of starch
played an important role. The formation of acids and of re
ducing sugars from starch cannot be relied upon to indicate
hydrolysis. Iodine color tests were much better for this
purpose, although false positive tests are possible. In the
case of negative results, i.e. starch not attacked, several
tests should be conducted simultaneously.
The formation of amylase by stenothermophilic bacteria
may be enhanced by (a) selecting a suitable protein medium,
(b) keeping the concentration of starch low, and (c) incubat
ing the strains sufficiently long, under cultural conditions
appropriate for the organism.
(c) Cell-free, starch saccharifying amylase preparations
were obtained from the medium of B. stearothermophilus ATCC
#7954 at 65 and 70 C. The enzyme preparation hydrolyzed
starch strongly, dextrin weakly and maltose not at all. It
was free from maltase, hut may have contained a glucosidase
acting at pH 11.
Saccharification of soluble starch proceeded slowly over
a wide range of pH and of temperature. Activity above 60 C
was better than that below. Optimum temperature was between
65 and 75 0. The optimum pH was between 5 and 7. Better
activity was noted under acid conditions and was still very
high at pH 3. The limits of the conversion of starch to re
ducing substances, calculated as maltose, were between 52
and 58 per cent of the total theoretically possible amount
of maltose with cell-free amylase, and between 55 and 77 per
cent in the presence of cells. Cells elaborated probably
maltase in addition to amylase. For working purposes, 60 per
cent were arbitrarily selected.
Sorensen’s phosphate buffer, less suitable than universal
buffer (monosodium phosphate, boric acid, acetic acid), de
pressed activity of the cell-free enzyme. Calcium carbonate
had the same effect in the medium during the first 24 hours
of incubation. Also, it failed to neutralize the acids
formed.
Different sources of errors in the experimental pro
cedures were noted and discussed in detail.
ACKNOWLEDGEMENTS
The advice, encouragement and kind understanding
of Doctor Philip A. Tetrault, Professor of Bacteriology,
are acknowledged with deep gratitude. His wise counsel,
freely given in difficult moments, contributed much to
the successful completion of this thesis.
I wish to thank Mr.K.S.Read for preparing specimens
for the electron microscope and for photographing their
flagella.
The financial assistance of the Purdue Research
Foundation is gratefully acknowledged.
table of contents
Page
INTRODUCTION 1
REVIEW OF THE LITERATURE 4
The Dissimilation of Starch and of Sugars by ther
mophilic Bacteria by C. Coolhaas 4
Thermophilic Amylolytic Bacteria by Imsenecki,
Solntzewa and Kuzyurina 8
The Production of Amylase Preparations from the
Culture of thermophilic Bacteria by Imsenecki
and Solntzewa 12
Adsorption of bacterial Enzymes by Chalk by Imse
necki and Avdievich 14
PART I. A DETERMINATIVE STUDY OF AMYLOLYTIC, STENO
THERMOPHILIC BACTERIA ISOLATED FROM SOIL 1?
INTRODUCTION 18
ISOLATION OF CULTURES 20
First Group 20
Second Group 22
PURIFICATION AND CARE OF CULTURES 25
MATERIALS AND METHODS 2?
DESCRIPTION OF CULTURES 31
DISCUSSION 57
SUMMARY OF PART I. 67
PART II. CULTURAL CONDITIONS AND THE BREAKDOWN OF
STARCH BY STENOTHERMOPHILIC BACTERIA 68
INTRODUCTION 69
