MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY TRAN THANH TRUC ISOLATION AND SCREENING OF ASPERGILLUS NIGER BIOSYNTHESIZE HIGH ACTIVITY PECTIN METHYLESTERASE DISSERTATION OF DO
Trang 1MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
TRAN THANH TRUC
ISOLATION AND SCREENING OF
ASPERGILLUS NIGER BIOSYNTHESIZE
HIGH ACTIVITY PECTIN METHYLESTERASE
DISSERTATION OF DOCTORAL DEGREE IN BIOLOGY
Specialization: Microbiology
Field of Study Code: 62 42 01 07
Supervisors
Assoc Prof Dr NGUYEN VAN MUOI
Dr NGUYEN VAN THANH
Can Tho, 2013
Trang 2SUMMARY
The thesis “Isolation and screening of Aspergillus niger biosynthesize high
activity pectin methylesterase” was performed in the laboratory of Food Technology Department, College of Agriculture and Applied Biology, Can Tho University and other laboratories, from 11.2008 to 9.2012
The thesis was conducted to investigate the biosynthesis process and application of pectin methylesterase (PME, EC 3.1.1.11) in solid state
fermentation (SSF) of isolated and selected Aspergillus niger from the
practical conditions of Vietnam
Sixty species which have morphologically similar Aspergillus niger were
isolated from the peels of rich pectin fruits which have been found to be widely in Mekong delta, such as orange, pomelo, citrus, apple and fig peels
on Potato dextrose agar (PDA), Czapek Dox Agar (CZ) and Malt Extract Agar (MEA) To indicate the pectinase activity of the organisms, diameters of clear zone around colonies on pectin agar medium were measured The result showed that all of sixty isolates have the ability of pectinolytic activities, in which 14 out of the 60 isolates were able to produce a higher pectinase activity, based on the average clear zone diameter were larger than 20 mm Further screening of PME production of the 14 selected isolates, the maximum PME production was obtained by 4 isolates (T1, N1, So2 and R1) which were
isolated from the peels of Ziziphus mauritiana (“Tao ta”), Citrus lemon (“chanh num”), Citrus sinensis L (“cam soan”) and Citrus maxima (“Nam Roi” pomelo), respectively The combination of two isolates
of R1 and So2 with the proportion of 1:1, was conducted to investigate the suitable conditions for PME biosynthesis by solid state fermentation
In addition, the identification by sequencing of 28S rRNA gene, revealed that
4 selected isolates T1, N1, So2 and R1 had 99 ÷ 100% identity to Aspergillus niger
The optimal conditions of pectin methylesterase biosynthesis from
Aspergillus niger So2 and R1 in solid-state fermentation were evaluated, based
on the individual factors (substrate characteristics, nutrients, buffer solutions and incubation time) which influenced to PME production were investigated After that, the Response surface methodology (RSM) based on four-variable central composite design (CCD) can be used to model the correlation of the fermentation conditions such as inducer concentration, initial pH, incubation temperature & moisture content of medium to PME activity Moreover, some
Trang 3extraction parameters, consist of type of solvent, pH, ratio of solvent and fermented solids were studied In addition, the best combination of extraction temperature, contact time was also determined
Especially, the use of rich pectin of agricultural material in Mekong Delta (mixture of apple pomace and pomelo peels at the ratio of 1:1 w/w) as a fermentation medium was taken into consideration for the PME production
of A niger in this investigation With the addition of 0.1% urea combined
with 0.5% MgCl2 and 0.15% CaCl2 into fermentation media, the highest PME obtained after 96 hours incubation at a temperature of 35.5°C and pH 4.0 (adjusted by citrate buffer), fermentation temperature and moisture content adjusted to 57.4% and 16.5% v/w of inducer concentration (105 cfu/mL) In addition, citrate buffer (pH 3.6) at 35°C and 50 min provided the best recovery in order to obtain highest value of PME The ratio 2:1 (v/w) of solvent and substrate was determined as an optimal condition for concentrated enzyme extracts Maximum production of PME (about 79.1 U/g
of substrate) was recorded under optimum conditions of SSF and extraction Ethanol was the appropriate precipitant for PME purification with the optimal ratio of cold ethanol and crude enzyme 3 : 1 (v/v) In this case, specific activity of PME was 10.02 U/mgprotein, the purification factor achieved 7.98 fold and the PME recovery yield obtained 91.48% Combined with the cross flow membrane step, the specific activity of PME reached to 15.63
comparison to the crude enzyme solution The molecular weight of the
enzyme was found to be 43 kDa and Km was 0.6014 mg/mL The activity of
the enzyme was stimulated by NaCl or CaCl2 In addition, the optimum
temperature and pH of A niger PME were 38 ÷ 40°C and 5.0, respectively Isothermal inactivation of partial purified A niger PME could be described by
a fractional – conversion model and the inactivation rate constants increase with increasing temperatures from 50 to 70°C
The obtained technical PME enzyme also proved the positive effects to improving of the texture of frozen pineapples and fermented cucumbers
Key words: Aspergillus niger, isolation, high activity, pectin methylesterase, rich pectin substrate, screening, solid state fermentation, texture
improvement.