Summary and conclusion
“The first requisite for success is the ability to apply your physical and mental
energies to one problem constantly without growing weary”
- Thomas A. Edison
V
Summary and conclusion
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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Summary and conclusion
5. Summary and conclusion
The bacterial sources have been exploited as a major source of melanin with
potential commercial applications in the field of cosmetics, pharmaceuticals and
agriculture (Zhang et al. 2007, Riley 1997), hence its optimization is important for
large scale production. However very few reports available on bacterial melanin
production as compared to fungal melanin and most of these paying attention only on
isolation, purification and characterization of melanin but not on optimization of
media conditions. (Dastager et al. 2006, Plonka and Grabacka 2006, Sajjan et al.
2010). Therefore improving melanin production from bacterial species is of great
interest for commercial applications. Among the microbial strains screened, a Bacillus
sp. JPJ used in this investigation has a competence to produce melanin using
L-tyrosine as a precursor and has the possibility of being used as a commercial source
for the large industrial scale production. Because of high production cost and its high
commercial value has given rise need of a demanding research for cheaper production
methods for the melanin. Physical parameters and nutritional requirements play a vital
role in the production of melanin from bacterial sources (Lagunas-Munoz et al. 2006),
so these parameters were optimized in the current study.
In recent years, L-DOPA production has attracted considerable research
interest because of its potential in treatment against Parkinson’s disease. Although
L-DOPA was produced by various methods, very little research was focused on
detailed optimization. Therefore in the present study optimization of process for the
maximum L-DOPA was carried out by evaluating various media components and
physical conditions.
The thesis focused on isolation of potential melanin producing bacteria and
optimization of physical and nutritional parameters for the highest melanin
production. The second major part of the thesis included the optimization of physical
parameters and evaluation of various carbon sources, nitrogen sources, influence of
mineral salts and vitamins for the maximum L-DOPA production. The enzyme system
involved in both L-DOPA and melanin production was studied.
The optimization of process conditions and media components resulted in
highest melanin yield by isolated bacterium Bacillus sp. JPJ produces within shortest
incubation period. The extensive literature survey reveled that this is the first report
on detailed optimization and the highest production of melanin by using bacterium
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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Summary and conclusion
Bacillus sp. JPJ within shortest incubation time which was very valuable for industrial
scale production of melanin. The optimum medium composition included 1.5 g l-1
sucrose, 2 g l-1 tryptone, 1.5 g l-1 Beef extract, 0.5 g l-1 L- ornithine, 0.2 g l-1 L-DOPA,
0.04 g l-1 CuSO4, 0.002 thiamine g l-1, 2 g l-1 L-tyrosine with pH 6, and flasks were
incubated at 30° with agitation rate 120 rpm. The scale up of melanin production with
6% inoculum size, 300 rpm agitation rate and 40% DO resulted in yield of 1.972 g l-1
of melanin within 48 h. These optimized conditions for scale up along with
intermittent addition of 2 g l-1 L-tyrosine with 6 h of time interval from 12 h to 30 h,
rather than only an initial concentration of 2 g l-1 resulted in the highest yield of
melanin (9.710 g l -1) within 52 h. The medium after incubation was showed dark
brown colored melanin pigment. The produced melanin was confirmed for its purity
by using spectroscopic analysis such as UV-Vis spectroscopy, FTIR and EPR. The
extensive literature survey reveled that this is the first report on detailed optimization
and the highest production of melanin by using bacterium Bacillus sp. JPJ within
shortest incubation time which was very valuable for industrial scale production of
melanin.
To study the effect of inducers on melanin production, well known inducer of
tyrosinase was evaluated which includes CuSO4 (Shrishailnath et al., 2010). This
resulted in increased melanin production of 0.792 g l-1 at optimum 0.04 g l-1 CuSO4.
The CuSO4 enhanced the melanin yield, because tyrosinase is a copper containing
enzyme (Clus and Deaker, 2006).
The inhibitors of melanin synthesis were valuable in cosmetic products to
increase the fairness of skin. Thus the effect of well known inhibitors such as
L-ascorbic acid and kojic acid was studied on bacterial melanin production. The effect
of L-ascorbic acid showed that, melanin production decreased suddenly from 1.928 g
l-1 to 0.384 g l-1 with 0.06 g l-1 of L-ascorbic acid. The kojic acid at the concentrations
of 0.08 g l-1 inhibits the melanin production from 1.928 g l-1 to 0.432 g l-1. The effect
of medicinal plants extracts was studied on melanin production which demonstrated
that melanin synthesis strongly inhibited by citrus fruit juice from 1.928 g l-1 to 0.028
g l-1 ; it was followed by saffron extracts and turmeric powder extract by producing
0.103 g l-1 and 0.267 g l-1 of melanin respectively. Our finding supports the use of
saffron, turmeric and almond for skin fairness as described in ‘Ayurveda’.
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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Summary and conclusion
The comparative melanin production by two strains indicated that, the highest
melanin yield of 9.710 g l-1 was obtained from Bacillus sp. JPJ while Brevundimonas
sp. SGJ produced less melanin of 8.931 g l-1. The incubation period required for
Brevundimonas sp. SGJ was 60 h, as compared to this starins Bacillus sp. JPJ was
observed to produce highest melanin within short incubation period of 52 h.
The new melanin production technique was developed in this study, which
involved biological transformation of L-tyrosine to melanin was carried out in a
buffer containing L-tyrosine and cell mass of bacterial strain Bacillus sp. JPJ. This is
the first report of fastest melanin production by using such a technique. The highest
melanin production of 1.24 g l-1 (82.66%) within 3 h of incubation period. The
optimized reaction conditions were pH 7, 1 g l-1cell mass, 0.04 g l-1 CuSO4, 1.5 g l-1
L-tyrosine and incubated at 30°C. The repeated use of the cell mass resulted in the
total melanin yield of 3.962 g l-1. The melanin production was confirmed by chemical
characterization and analytical studies like FTIR and EPR.
The ability of Bacillus sp. JPJ to produce L-DOPA efficiently was investigated
in this study. The optimization physical and nutritional requirements for L-DOPA
production was carried out which resulted in highest yield L-DOPA. The optimized
medium conditions included 1.5 g l-1 sucrose, 2 g l-1 tryptone, 1.5 g l-1 beef extract,
0.04 g l-1 CuSO4, 0.02 g l-1 L-ascorbic acid and 2 g l-1 L-tyrosine, with pH 6, and
incubated at 30° with agitation rate 120 rpm. This is the first report of highest
L-DOPA production using bacterial system. The scale up of L-DOPA production was
carried out using lab scale fermenter resulted in the highest yield of 11.138 g l-1
L-DOPA by consuming 11.910 g l-1 L-tyrosine within 48 h. The optimum parameters
for scale up observed were inoculum size of 6%, 300 rpm agitation rate with 40%
dissolved oxygen. These optimized conditions for scale up along with intermittent
addition of 2 g l-1 L-tyrosine with 6 h of time interval from 6 h to 30 h, rather than
only initial concentration of 2 g l-1 resulted in the highest L-DOPA yield of 11.738 g l-1
by consuming 11.910 of L-tyrosine within 48 h. The L-DOPA production was further
confirmed by HPTLC, HPLC and GC-MS.
The use of tyrosinase inhibitors to enhance the L-DOPA yield showed that,
among the standard inhibitors L-ascorbic acid increased the L-DOPA production. The
use of natural inhibitors such as citrus fruit juice proved that it is cost effective
alternative for standard ascorbic acid to enhance the L-DOPA yield.
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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Summary and conclusion
The comparative L-DOPA production by two strains showed that, the highest
L-DOPA yield of 11.138 g l-1
was obtained from Bacillus sp. JPJ while
Brevundimonas sp. SGJ by producied less L-DOPA of 9.245 g l-1. The incubation
period required for Brevundimonas sp. SGJ was 54 h, as compared to this starins
Bacillus sp. JPJ was observed to produce highest melanin within short incubation
period of 48 h.
The production of L-DOPA by alternative method in a buffer added with
L-tyrosine and Bacillus sp. JPJ was optimized in the current study. The L-DOPA
production was achieved by this method was 0.497 g l-1 (99.4 %) by utilizing 0.500
mg ml-1 (100%) of L-tyrosine within 60 minutes of incubation period. The optimized
reaction conditions used were pH 8, temperature 40°C, 1 g l-1 cell mass, 0.5 g l-1
L-tyrosine, 0.06
g l-1 CuSO4, 0.04 g l-1 L-ascorbic acid and 2 g l-1 activated charcoal.
This is the first report of L-DOPA production in a buffer using bacterial system. The
repeated use of the cell mass resulted in the total L-DOPA yield of 1.613 g l-1. The
bacterial system used here is efficient and superior over the earlier sources because of
less incubation period, less cell mass and requirement of simple media components.
Our bacterial isolate Bacillus sp. JPJ was found to have the potential to
produce melanin and L-DOPA. Since synthesis of melanin involves the enzyme
tyrosinase in the initial steps of reaction for the conversion of L-tyrosine to L-DOPA
this enzyme was selected for further purification and primary characterization.
The tyrosinase was purified by ion exchange chromatography using DEAE
cellulose column. The enzyme was eluted at the eluent (0.1 M NaCl) showing 8071 U
mg-1 specific activity with a 2.51 purification fold and 34.97 % yield. The molecular
weight of the purified tyrosinase was estimated by polyacrylamide gel electrophoresis
was near about 34 kDa. The activity staining by using L-DOPA confirmed the protein
was tyrosinase. The characterization of enzyme resulted in optimum pH 9 and
temperature 30°C with tyrosinase activity of 8240 U mg-1. Substrate specificity was
determined using three substrates, among these substrates the enzyme showed higher
specific activity towards catechol with activity of 8071 U mg-1 than L-DOPA (7128
U mg-1) and L-tyrosine (5481 U mg-1). The effect of 7 metal ions was tested in the
present study which indicates that the activity of tyrosinase enhanced to 9157 U mg-1
in presence of CuCl2 and it was inhibited by HgCl2 (149 U mg-1). The kinetic
characterization of tyrosinase was performed using Lineweaver-Burk plot. The Km
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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Summary and conclusion
and Vmax values for tyrosinase were found to be 0.649 mM and 8116 U mg-1
respectively.
In conclusion Bacillus sp. JPJ present themselves as a promising new source
for bacterial melanin and L-DOPA production which possess superior distinctiveness
over earlier sources, in turn facilitate highest melanin and L-DOPA yield within short
period, and proves its economic feasibility and ability for the large scale production
of melanin and L-DOPA.
A novel approach for biotransformation of L- tyrosine to melanin by microbial system
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