Effect of Yeast Extract On Alpha-Amylase Synthesis by Bacillus Amyloliquefaciens
Effect of Yeast Extract On Alpha-Amylase Synthesis by Bacillus Amyloliquefaciens
Effect of Yeast Extract On Alpha-Amylase Synthesis by Bacillus Amyloliquefaciens
TIME IN HOURS
Figure 1. Effect of yeast extract on a-amylase fermentation by B . urnyloliquefu-
ciens. Open and filled symbols represents fermentation in 1 and 5 g/L yeast extract,
respectively (see text for medium composition). Symbols: a-amylase fV,V); biomass
(A& glucose (El,=); pH (0,O).
2
'X
200
. V
175 -
150 -
v\
125 - \
I00 -
\v . W
75 -
i
GLUCOSE CONCENTRATION, g/l
Figure 2. Change in overall productivity with increasing glucose concentration
in batch fermentation with 1 g/L yeast extract.
5 g/L keeping the glucose concentration constant around thesis but may stimulate a-amylase synthesis .' Commer-
19 g/L. Figure 1 represents a typical batch fermentation cially complex nitrogenous sources are used to sustain
data with 1 and 5 g/L concentration of yeast extract in the synthesis of both a-amylase and pr~tease.~." A complica-
medium. In presence of 5 g/L yeast extract the a-amylase tion concerning the effect of complex nitrogenous sources
activity in the medium was found to be extremely low is the likelihood of a-amylase destruction by inducible
(hereafter to be referred to as the nonproducing system). pro tease^.^ The protease synthesis characteristics were
Analysis of batch data with 1-5 g/L yeast extract concen- thus studied in a medium containing 1 g/L yeast extract
tration in the fermentation medium shows that the overall at a constant pH of 7.0 (in the New Brunswick fermentor).
productivity of fermentation initially increased slightly be- The protease production was found to be concomitant with
tween 1 and 2 g/L yeast extract concentration and then fell the a-amylase synthesis but reached a plateau in 10 h
very rapidly beyond this point. The results are shown in compared to 28 for a-amylase. About 2000 DAPU/mL
Figure 3. To further investigate if the cells had lost their alkaline protease and 6500 LAU/ml a-amylase were syn-
ability to produce a-amylase in the medium containing thesized from 10 g/L glucose. The a-amylase produced
yeast extract at 5 g/L, a 10-mL sample was withdrawn during the fermentation may be degraded by this protease.
from the fermentor after 18 h (shown by the arrow in The possibilities of the protease degradation of a-amylase
Fig. 1) and was incubated in a medium containing 1 g/L will be discussed later.
yeast extract without any glucose. In 12 h, the cell concen- The effect of enzyme deactivation in the fermentation
tration increased by 164%, with enzyme production level systems were investigated next. The strain B. amylolique-
reaching 1647 LAU/mL. This indicates that the cells faciens is known to produce considerable amounts of 2,3-
grown in the medium of yeast extract at 5 g/L were not butanediol, acetate, and lactate.I4 Under uncontrolled pH
physiologically different from the cells grown in the experiments, increasing the yeast extract concentration re-
medium containing yeast extract of 1 g/L. sults in increased acid production. The total acid produc-
Bacillus amyloliquefaciens F is known to produce tion in 5 g/L yeast extract fermentation was 4 times higher
mostly alkaline protease in addition to a-amylase and in the uncontrolled pH system over the same fermentation
small amounts of neutral p r ~ t e a s e . The
~ . ~ activity ratio of at a constant pH of 7. The levels of acid and solvent pro-
alkaline protease to neutral protease produced by the duced in various experiments are presented in Table I.
present organism was reported to be over 111.6 It is well These metabolites are likely to deactivate the a-amylase
established that free amino acids repress protease biosyn- produced in the system. Hence, deactivation of a-amylase
iin
100:
90 :
80 :
70 :
60 :
50 :
40 :
30 :
20 7 v\ V
10 r
\
was studied in the cell-free fermentation broth (systems A , the cell-free broth from the 5 g/L yeast-extract-containing
B, and C) as well as fresh medium (system D). Details of system (system C) was higher compared to the deacti-
these systems are given in the Materials and Methods vation in the broth from 1 g/L yeast extract fermenta-
section. The results of the stability tests are presented in tion (system A), it is obvious that it cannot account for
Figure 4. It appears from Figure 4 that the extent of de- lower levels of a-amylase synthesis in high-yeast-extract-
activation in 25 h was between 20 and 30% in all four dif- containing systems ( 5 g/L yeast extract).
ferent systems studied, indicating that the deactivation of The mode of glucose utilization in the a-amylase pro-
a-amylase by the metabolites cannot account for the al- ducing and nonproducing systems was essentially the
most zero level of a-amylase in the medium with yeast ex- same, except the presence of additional yeast extract in the
tract at 5 g/L. It also indicates that the degradation of nonproducing system supported a somewhat higher growth
a-amylase by protease produced in the medium containing rate. The higher growth rate with increasing yeast extract
yeast extract at 5 g/L (systems B and C) was not signifi- concentration was associated with a more rapid fall in the
cant. Even though the rate of deactivation of a-amylase in pH of the system, as shown in Figure 1. The decrease in
pH was the result of high acid concentration in uncon-
Table I. Production levels of acids and solvents." trolled conditions. For this reason we further investigated
Yeast extract Hours of Acetic acid Butanediol the effect of keeping the pH constant throughout the fer-
(g/U PH fermentation (g/L) (g/L) mentation (5 g/L yeast extract). The fermentation data is
~
m
0
m
0
4
(D
03
(D
CONCLUSIONS 2. G. Coleman, J . Gen. Micrbioi., 49, 421 (1967).
3. N. E. Welker and L. L. Campbell, J . Bacteriol., 86, 681 (1963).
In previous studies yeast extract was found to be essen- 4. G. Coleman and M. A. Grant, Nature. 211, 306 (1966).
tial to obtain a good growth of B . amyloliquefaciens. Our 5. Q. Zhang, N. Tsukagohi, S . Miyashiro, and S . Udeka, Appl. Envi-
studies indicate that there is an optimum yeast extract con- ron. Micrbiol., 46, 293 (1983).
6. M. B. Ingle and E. W. Boyer, in Microbiology, D. Schlessinger,
centration for a-amylase production. For uncontrolled pH
Ed.,Washington D.C. (American Society of Microbiology, 1975),
fermentations, increasing the yeast extract concentration to p. 420.
a level of 5 g/L lowers the pH significantly. This results in 7. W. M. Fogarty and C. T. Kelly, in Microbial Enzymes and Biocon-
the complete repression of the enzyme synthesis. Under versions, A. H. Rose, Ed. (Academic, New York, 1980), p. 115.
controlled pH condition, the a-amylase synthesis with the 8. B. K. May and W. H. Elliot, Biochem. Biophys. Acta, 157, 607
(1968).
medium containing yeast extract at 5 g/L was comparable
9. Y. J. Yoo, Ph.D. Dissertation, “Kinetics and Optimal Control of
to that with yeast extract at 1 g/L. This study suggests that a-amylase Production from Bacillus arnyloliquefaciens” University
a strict pH control is required in complex media containing of Maryland, Maryland, 1986.
high levels of yeast extract for studying the a-amylase syn- 10. L. Nyiri, Inr. Chem. Eng., 11, 447 (1971).
thesis by B . amyloliquefaciens. 1 1 . F. G. Heineken and R. J. O’Connor, J . Gen. Microbiol., 73, 35
(1972).
The authors would like to thank Mr. John Ponzo for valuable dis-
12. A. C. Blackwood, A. C. Neish, W. E. Brown, and G. A. Ledingham,
cussion on culture maintenance.
Can. J . Res., 25B, 56 (1947).
13. E. T. Papoutsakis and C. L. Meyer, Biotechnol. Bioeng., 27, 50
(1985).
References 14. S. Alam, Ph.D. Dissertation, “Fermentation and Separation in Aque-
ous Two-Phase System,” Illinois Institute of Technology, Chicago,
1. T. Maniatis, E. F. Fritsch, and J. Sambmok, Molecrrtar Cloning,Cold IL, i988.
Spring Harbor, N.Y., (Cold Spring Harbor Laboratory, 1982). 15. R. J. Magee and N. Kosaric, Adv. Appl. Microbiol, 32, 89 (1987).