Abstract:
In the present study, eighty six strains of Saccharomyces cerevisiae were isolated
from different samples of fruits and soil by serial dilution method. The strain IS-66 gave
maximum extracellular invertase production (1.10 U/ml). The enzyme activity reached to
5.6 U/ml when incubation time (48 h), sucrose concentration (5 g/l) and pH 5.5 were
optimized. The wild strain IS-66 was exposed to ultraviolet (UV) radiations to obtain a
mutant with improved enzyme activity. UV induced mutagenesis did not produce any
stable mutant and almost all of the mutants produced relatively lesser invertase than the
parental strain. Strain IS-66 was further subjected to chemical mutagenesis using nitrous
acid and ethyl methane sulphonate (EMS). After extensive screening, two mutants were
developed with increased enzyme activity NA-45 (20.74 U/ml) and EMS-42 (34.2 U/ml)
from the wild-culture (IS-66). The mutant strain EMS-42 was cultured on the medium
containing 2-deoxy-D-glucose (2dg) and its stability in invertase production was
determined at different concentrations of 2dg. The concentration of 0.04 mg/ml was
found optimal, as at this concentration EMS-42 showed consistent enzyme activity.
Six media were evaluated for the production of invertase in shake flasks. M1
medium (g/l) containing yeast extract 3, peptone 5 and sucrose 30 g/l gave better
production of invertase (25.28 U/ml) after 48 h of inoculation. Different sugars such as
sucrose, glucose, fructose, lactose, galactose, maltose, raffinose and molasses were
investigated on the enzyme production. Of these, sucrose was found to be best (44.03
U/ml) after optimizing the concentration at 10 g/l. Incubation temperature (30oC),
inoculum size (2.0 %, v/v) and volume of the medium (50 ml/250 ml Erlenmeyer flask)
were optimized. The effect of different additional nitrogen sources such as organic,
inorganic and agriculture byproducts were also tested. Peptone at the concentration of 6
g/l gave maximum production of invertase (50 U/ml). The addition of inorganic nitrogen
sources and agricultural byproducts nitrogen were not found to have any impact on the
enhancement in enzyme production rather it was decreased from the control especially
in case of agricultural byproducts. In stirred fermentor (7.5 L), the scale up studies for
invertase production was carried out. The enzyme production (65.12 U/ml) was obtained
after 24 h of incubation. The overall increase in enzyme activity (15 U/ml) and
fermentation time was shortened by 24 h while scaling up enzyme production from shake
flask to fermentor. The maximum enzyme activity (80.06 U/ml) was achieved after
xvioptimization of cultural conditions such as sucrose (15 %, w/v), pH (4.5), inoculum size
(7.5 %, v/v), agitation intensity (240 rpm) and aeration rate (1 vvm, 10 % DO).
The intracellular enzyme activity was also determined by sonication. The
maximium enzyme activity (57 U/ml) was found in mutant strain of EMS-42 after 24 h
fermentation in the fermentor. During sonication, the maximum specific activity of 106
U/mg of protein was obtained with 0.5 duty cycle of impulses at amplitude of 40 % and
pH 5 for 60 min. The calcium alginate entrapment technique was used for
immobilization of whole cells of S. cerevisiae EMS-42 to form inverted syrup. It was
noticed that maximum sucrose hydrolysis (63.40 %) was achieved after 18 h of
incubation time. By optimization of cultural conditions for sucrose hydrolysis, the
maximum hydrolysis percentage (76.3 %) was obtained at 50oC, pH 5.5 using sucrose
(60 %) as substrate.
An extracellular invertase was purified to homogeneity by two step purification
i.e., ammonium sulfate precipitation and DEAE-Sephadex A-50. The enzyme was
present in the supernatant of 85 % saturation being glycoprotein in nature. DEAE
column chromatography eluted the enzyme as single active fraction at 0.2 M NaCI. The
enzyme was purified by 15 fold with recovery of 38 %. The molecular mass of 110 kDa
was determined after SDS-PAGE. The carbohydrate content was found to be 48 %. The
intracellular invertase contains both forms of glycosylated (large) and non-glycosylated
(small). The same procedure was applied for glycosylated intracellular invertase (L-
form) while three purification steps were performed for non-glycosylated invertase (S-
form). The L-form was purified by 19 fold with recovery of 32 %. Like extracellular
invertase, the molecular weight (110 kDa) for L-form was found. Ammonium sulfate
precipitation separated the enzyme (S-form) as insoluble fraction. This form of enzyme
was eluted at 0.3 M NaCl using DEAE-Sephadex. A single band of molecular weight (55
kDa) was estimated after Sephadex G-50 with purification (16 fold) and recovery of 17
%. For both purified non-glycosylated and glycosylated invertase the optimum pH (5)
was same whereas optimal temperature, MnCl 2 and the values of the K m and V max were
found to be as 50 and 60oC, 109 and 111 %, 1.2 and 1.8 mM, 909 and 1429 U/ml/min,
respectively.