Seminar on Graduate Institute of Food Science and Biotechnology of National Chung Hsing University
Production, purification and characterization of two α-amylase
isoforms from a newly isolated Aspergillus Oryzae strain S2
Speaker
Moderator
：Ling Hung Ho (林訓豪, 7101043041)
：Yi-Chung Fu, Ph D
1. Introduction
Amylases, known as enzyme which can catalyze the hydrolysis of the α(1-4)
glucosidic linkage of polysaccharides. Aspergillus oryzae is non-toxigenic and
non-pathogenic microorganisms, and has been reported to produce several amylases.
They are widely used in traditional fermentation industries, including soy sauce, sake,
bean curd seasoning, miso, shochu, and vinegar production.
In this study, a new fungal strain was isolated from old sweet soy sause was
indentified. The latter was noted to produce two extracellular α-amylases called
AmyA and AmyB. The molecular masses estimated for both amylases by SDS-PAGE
were 50 and 42kDa, and attained maximal activity at pH5.6 and 50℃. Major starch
hydrolysis end products were maltose and maltotriose, they were also activated and
stabilized by Ca2+.
2. Materials and methods
1. Fungal isolation and identification
Sixty new fungi were isolated from an expired sweet soy sauce which
cultured on Czapek medium. Subculturing until pure isolated were obtained,
selects α-amylase producing colonies by flooding the plated with iodine
2.
3.
solutions. To verify the species of the S2 strain, different aflatoxins assays
were performed.
Amylase production
Using the optimized medium for α-amylase production for Aspergillus
oryzae culture, yield of 350 Uml-1 after 92h. In the absence of protease
inhibitors, AmyB was also noted to increase.
Purification of amylases
The crude extract of 24 and 100h old culture were used for the purification
of AmyA and AmyB. Involve the application of the old crude extract to size
exclusion chromatography. The fraction containing amylase activity was
applied to FPLC anion exchange as a last purification step
4.
Biochemical characterization of AmyA and AmyB
pH
Temp
: Both amylases with optimal activity between pH 5 and 6,
maxima at pH 5.6.
: Both enzymes have an optimum temperature of 50℃, and
retaining 80% of their activities in range of 40-55℃,
rapid decrease in activity beyond 60℃.
Metal ion : The enzymes partially inhibited by Mg2+, Fe2+ and Ba2+
(retaining more than 50% of activity), strongly inhibited by
Cu2+, Co2+, Mo2+ and Mn2+ (4mM). However, Ca2+ was noted to
activating and stabilizing ion.
3. Results and discussion
In the absence and presence of protease inhibitors, strongly suggested that AmyB
was a derivative of AmyA following a proteolytic degradation (50 kDa degrade to
42kDa). However, AmyB isoform whose specific activity of 6686.3 Umg-1 was
higher than AmyA (5089.3 Umg-1).
In the analysis of NH2-terminal sequences showed that both isoforms had similar
amino acid sequences. The optimum temperature and pH of stability and activity of
isoenzymes were globally similar and in consistency with the ones previously
reported for other Aspergillus α-amylases. The enhancement of amylase activity by
Ca2+ ions can be attributed to their ability to interact with negatively charged amino
acid residues, such as aspartic and glutamic acids.
The starch hydrolysis products of AmyA and AmyB were analyzed by HPLC,
maltose and maltotriose were the main end products. So it can be concluded that these
amylases are endoamylases belonging to the α-1, 4-glucan-4-glucanogydrolase.
References
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