biologia - Studia

CHARACTERIZATION OF ALPHA-AMYLASE FROM BACILLUS SUBTILIS STRAIN
important enzymes have traditionally been produced by submerged fermentation,
but in recent years slide-state fermentation processes have been increasingly used
for the production of these enzymes. In fact, solide-state fermentation has gained
renewed interest from researches for the production of these enzymes in view of its
economic and engineering advantages (Pandely, 1992). Inexpensive agriculture and
agro-industrial residues are generally considered the best substrates for solid-state
fermentation and for enzyme production in the systems (Pandely, 2003).
Our research aims at obtaining a valuable strain of Bacillus subtilis for
producing alpha-amylase, using the UV-irradiation technique. We have also tested
an enzymatic preparation of alpha-amylase under laboratory conditions, followed
by partial purification and enzyme kinetics studies of alpha-amylase.
Materials and methods
Microbiological analyses. The Bacillus subtilis strain with amylolytic capacity
was obtained by using a culture medium made of nutritive agar to which soluble
starch was added to 0.5 %. There were used for inoculation several sources of
microorganisms (fertile soils, therapeutic mud, aquatic sediments, compost). There
were selected 5 bacterial strains with an intense amylolytic activity (that
hydrolysed all the starch from the solution). There were obtained pure cultures and
the specific affiliation as Bacillus subtilis strains was determined by microscopic,
physiological and biochemical tests. The isolated strains were exposed to UV radiation
(250 nm), and then the amylolytic activity was tested again, both from qualitative and
quantitative point of view (by the Somogy-Nelson method) (Drăgan-Bularda, 2000).
Fermentation studies. There was used for fermentation a bubbling vessel
of 500 ml capacity. After the thermal sterilization, the vessel opening was sealed
with a rubber plug, provided with a sampling tube, so as to keep the aseptic
condition of the vessel. The vessel was placed in a water-bath with adjustable
temperature and connected to an adjustable-flow air-pump. In order to prevent the
contamination of the culture medium, the air supply was provided with a filtering
system with pores of 5, 2 and 1 µm. The discharged air was bubbled into a disinfecting
solution before its exhausting, in order to ensure aseptic conditions and to prevent
the release of culture microorganisms in the atmosphere.
Preparation of the culture medium. It had the following composition
(Atlas, 2004): soluble starch 2%, yeast extract 0.3 %, (NH 4 ) 2 SO 4 0.3%, K 2 HPO 4
0.1 %, MgSO 4 . 7 H 2 O 0.02%, NaCl 0.1 %, CaCl 2 . 2 H 2 O 0.008%. The medium was
sterilised at 110 0 C, for 30 minutes.
Preparation of inoculum. The Bacillus subtilis strain, growth on a solid
medium with starch was inoculated on a liquid nutritive medium with starch. The
incubation was made at 28 0 C, for 24 hours. Then, there were introduced 15 ml of
culture from the liquid medium into 100 ml of nutritive medium. This mixture was
kept for 4 hours at 28 0 C. Further on, it is added to the fermentation vessel, that
contains 385 ml nutritive medium. The inoculum was mixed with the medium in
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