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11 Enzymes
agarase. Here we look at the process of extraction of lipase from marine microorganisms. The general approach to commercial production of enzymes from microorganisms is to cultivate the microorganism in a medium which promotes the metabolism
of the desired enzyme. For this purpose, it is important to study the growth kinetics
of the microorganism in order to identify the growth phase during which it produces
the desired enzyme at optimal rate. Once the desired amount of enzyme is produced
by the microorganism, the rest of the process is then directed at recovering and
purifying the enzyme free of the microorganism. One of the challenges of sourcing
enzymes from microorganisms is the risk of contamination of the final product with
the microorganism which could be pathogenic, especially where the enzyme is used
in food or pharmaceutical products.
Enzyme production from microorganisms is illustrated here using the method
presented in the literature for the extraction of lipase from marine bacteria as a case
study. In the study by Sivasubramani et al. (2013), marine bacterium Pseudomonas
putida isolated from the Vellar estuary in the Cuddalore district, Tamil Nadu, Southern India, is used as the source of the lipase enzyme. Other lipase-producing marine
bacteria strain includes Bacillus spp. (Valsa et al. 2003) and marine Streptomyces
sp. (Yuan et al. 2016).
The temperature, pH, salinity and nutrient composition are critical parameters to
be controlled when culturing microorganisms for enzyme production. In this particular example, the optimal values for these parameters were a neutral pH, 35 °C and
a sodium hydroxide concentration of 0.6%. The nutrient in the medium consisted
of 1% dextrose and 0.2% yeast extract. Companies such as Sigma-Aldrich supply
nutrient media which are optimal for required microorganism culture. The culture
was incubated for a duration of 48 h.
First, the microorganisms are isolated from the water of the estuary. The sample
is then incubated in Tween 80 agar plates to isolate the lipase-producing microbes.
The optimal growth conditions of the most productive strain are then identified by
growing at different growth conditions and finding the optimum pH, temperature,
salinity, nutrient composition and duration. The bacteria strain is then grown in a
shake flask using the optimal parameters determined. For a particular microorganism
and desired enzyme, the optimal growth conditions will vary. The optimal conditions
will also vary for different strains; therefore, the processor must determine the optimal
growth conditions for a specific strain and target enzyme.
The inoculum is then introduced into a bioreactor in a ratio of 1:100 (volume of
inoculum to volume of bioreactor medium). The culture was grown over a duration of
48 h at the optimal conditions. At the end of the process, the medium was filtered. The
liquid filtrate was precipitated with ammonium sulfate followed by centrifugation
at 3000 rpm for 30 min at 4 °C. The solid precipitate is then dissolved in 0.05 M
Tris-HCl and then purified by dialysis. The enzyme activity of the purified protein
was 9.474 U/mg at the highest when tributyrin is used as the triglyceride substrate.
SDS-PAGE analysis showed that the partially purified protein sample containing
the enzyme derived using the method described had molecular weights of 34, 45
and 52 kDa, which are within the range of molecular weight of lipase. The actual
molecular weight of lipase has been reported to be lower, and others have reported
11 Enzymes
agarase. Here we look at the process of extraction of lipase from marine microorganisms. The general approach to commercial production of enzymes from microorganisms is to cultivate the microorganism in a medium which promotes the metabolism
of the desired enzyme. For this purpose, it is important to study the growth kinetics
of the microorganism in order to identify the growth phase during which it produces
the desired enzyme at optimal rate. Once the desired amount of enzyme is produced
by the microorganism, the rest of the process is then directed at recovering and
purifying the enzyme free of the microorganism. One of the challenges of sourcing
enzymes from microorganisms is the risk of contamination of the final product with
the microorganism which could be pathogenic, especially where the enzyme is used
in food or pharmaceutical products.
Enzyme production from microorganisms is illustrated here using the method
presented in the literature for the extraction of lipase from marine bacteria as a case
study. In the study by Sivasubramani et al. (2013), marine bacterium Pseudomonas
putida isolated from the Vellar estuary in the Cuddalore district, Tamil Nadu, Southern India, is used as the source of the lipase enzyme. Other lipase-producing marine
bacteria strain includes Bacillus spp. (Valsa et al. 2003) and marine Streptomyces
sp. (Yuan et al. 2016).
The temperature, pH, salinity and nutrient composition are critical parameters to
be controlled when culturing microorganisms for enzyme production. In this particular example, the optimal values for these parameters were a neutral pH, 35 °C and
a sodium hydroxide concentration of 0.6%. The nutrient in the medium consisted
of 1% dextrose and 0.2% yeast extract. Companies such as Sigma-Aldrich supply
nutrient media which are optimal for required microorganism culture. The culture
was incubated for a duration of 48 h.
First, the microorganisms are isolated from the water of the estuary. The sample
is then incubated in Tween 80 agar plates to isolate the lipase-producing microbes.
The optimal growth conditions of the most productive strain are then identified by
growing at different growth conditions and finding the optimum pH, temperature,
salinity, nutrient composition and duration. The bacteria strain is then grown in a
shake flask using the optimal parameters determined. For a particular microorganism
and desired enzyme, the optimal growth conditions will vary. The optimal conditions
will also vary for different strains; therefore, the processor must determine the optimal
growth conditions for a specific strain and target enzyme.
The inoculum is then introduced into a bioreactor in a ratio of 1:100 (volume of
inoculum to volume of bioreactor medium). The culture was grown over a duration of
48 h at the optimal conditions. At the end of the process, the medium was filtered. The
liquid filtrate was precipitated with ammonium sulfate followed by centrifugation
at 3000 rpm for 30 min at 4 °C. The solid precipitate is then dissolved in 0.05 M
Tris-HCl and then purified by dialysis. The enzyme activity of the purified protein
was 9.474 U/mg at the highest when tributyrin is used as the triglyceride substrate.
SDS-PAGE analysis showed that the partially purified protein sample containing
the enzyme derived using the method described had molecular weights of 34, 45
and 52 kDa, which are within the range of molecular weight of lipase. The actual
molecular weight of lipase has been reported to be lower, and others have reported