Protein function depends greatly on its structure. Based on this principle, it is vital to study the
protein structure in order to understand its function. This study attempts to build the predicted
model of lipase gene in Rhodococcus sp. NAM81 using homology modelling method. The
predicted structure was then used to investigate the function of protein through several
bioinformatic tools. The DNA sequence of lipase gene was obtained from the Rhodococcus sp.
NAM81 genome scaffold. Blastx analysis showed 100% identity to the target enzyme andthe
appropriate template for homology modelling was determined using Blastp analysis. The 3D
protein structure was built using two homology modelling software, EsyPred3D and Swiss
Model Server. Both structures built obtained LGScore of greater than 4, which means they are
extremely good models according to ProQ validation criteria. Both structures also satisfied the
Ramachandran plot structure validation analysis. The predicted structures were 100% matched
with each other when superimposed with DaliLite pairwise. This shows that both structure
validation servers agreed on the same model. Structure analysis using ProFunc had found seven
motifs and active sites that indicate similar function of this protein with other known proteins.
Thus, this study has successfully produced a good 3D protein structure for the target enzyme.
Bacterial based remediation of environmental toxicants is a promising innovative technology
for molybdenum pollution. To date, the enzyme responsible for molybdate reduction to Moblue
from bacteria show that the Michaelis-Menten constants varies by one order of magnitude.
It is important that the constants from newer enzyme sources be characterized so that a
comparison can be made. The aim of this study is to characterize kinetically the enzyme from a
previously isolated Mo-reducing bacterium; Bacillus pumilus strain Lbna. The maximum
activity of this enzyme occurred at pH 5.5 and in between 25 and 35 oC. The Km and Vmax of
NADH were 6.646 mM and 0.057 unit/mg enzyme, while the Km and Vmax of LPPM were 3.399
mM and 0.106 unit/mg enzyme. The results showed that the enzyme activity for Bacillus
pumilus strain Lbna were inhibited by all heavy metals used. Zinc, copper, silver, chromium,
cadmium and mercury all caused more than 50% inhibition to the Mo-reducing enzyme activity
with copper being the most potent with an almost complete inhibition of enzyme activity
observed.
Biosurfactants are surface active compounds and amphiphatic in nature which consist of
hydrophilic head and hydrophobic tail accumulating at the interphase of two immiscible liquid
with different polarity. A study was conducted to investigate the effectiveness of sunflower oil in
the production of rhamnolipids (RLs) by locally isolated Pseudomonas aeruginosa in shake flask
fermentation. In this process, four different fermentation treatments were done for seven days at
30°C and 180 rpm. Sampling was carried out in time intervals of 24 h followed by monitoring of
cell growth and biosurfactants production. Colorimetric Orcinol analysis was used for
determination of RLs concentrations (g/L). The RLs were studied for emulsification activity
using emulsification index (E24%) methods. In addition, oil displacement activity and thermal
stability were also studied (4-120°C). All treatments allow the growth of P. aeruginosa and the
utilization of sunflower oil as carbon source and glucose as growth initiator were observed to be
the best strategy for maximum RLs production. The maximum RLs production was achieved
after 120 h with 3.18 g/L of RLs. Diesel shows the highest emulsification activity among the
substrate tested ranging from 55.56% - 60.00%. The oil displacement activity was corresponding
to RLs concentration with stability up to 120°C (for 60 min). Therefore, from this research a
good potential of RLs that may provide good application for industry were produced.
The issue of heavy metal contamination and toxic xenobiotics has become a rapid global
concern. This has ensured that the bioremediation of these toxicants, which are being carried out
using novel microbes. A bacterium with the ability to reduce molybdenum has been isolated
from contaminated soils and identified as Serratia marcescens strain DR.Y10. The bacterium
reduced molybdenum (sodium molybdate) to molybdenum blue (Mo-blue) optimally at pHs of
between 6.0 and 6.5 and temperatures between 30°C and 37°C. Glucose was the best electron
donor for supporting molybdate reduction followed by sucrose, adonitol, mannose, maltose,
mannitol glycerol, salicin, myo-inositol, sorbitol and trehalose in descending order. Other
requirements include a phosphate concentration of 5 mM and a molybdate concentration of
between 10 and 30 mM. The absorption spectrum of the Mo-blue produced was similar to the
previously isolated Mo-reducing bacterium and closely resembles a reduced phosphomolybdate.
Molybdenum reduction was inhibited by Hg (ii), Ag (i), Cu (ii), and Cr (vi) at 78.9, 69.2, 59.5
and 40.1%, respectively. We also screen for the ability of the bacterium to use various organic
xenobiotics such as phenol, acrylamide, nicotinamide, acetamide, iodoacetamide, propionamide,
acetamide, sodium dodecyl sulfate (SDS) and diesel as electron donor sources for aiding
reduction. The bacterium was also able to grow using amides such as acrylamide, propionamide
and acetamide without molybdenum reduction. The unique ability of the bacterium to detoxify
many toxicants is much in demand, making this bacterium a vital means of bioremediation.
Pollution in the environment is deteriorating the ecology due to human activities in a large array
of industrial and agricultural sectors. Bioassay of polluted waters using bioluminescent bacterium
has been touted as one of the most economical, rapid and sensitive tests. The growth of the
bacterium on seawater medium exhibited a typical sigmoidal profile. To extract important growth
parameters useful for further modelling exercise, various primary growth models were utilized in
this study such as Modified Logistic, modified Gompertz, modified Richards, modified Schnute,
Baranyi-Roberts, von Bertalanffy, Huang and the Buchanan three-phase model. The best
performance was Huang model with the lowest value for RMSE, AICc and the highest value for
adjusted R2. The AF and BF values were also excellent for the model with their values were the
closest to 1.0. The Huang parameters, which include A or Y0 (bacterial growth lower asymptote),
μm (maximum specific bacterial growth rate), l (lag time) and Ymax (bacterial growth upper
asymptote) were 7.866 (95% confidence interval of 7.850 to 7.883), 0.329 (95% confidence
interval of 0.299 to 0.359), 1.543 (95% confidence interval of 1.303 to 1.784) and 8.511 (95%
confidence interval of 0.299 to 0.359).
The trend of global under-five child mortality rate showed a steady decrease at -3.14% per year
approaching the United Nations millennium development goals target (-4.0% reduction per year
from 1990-2015). This rate is usually inversely proportional to the nations GDP. However, the
progress in the recent years (2006-2012) in Malaysia has raised a disturbing concern with an
annual average increment of +0.81% per year. Thus, there is an urgent need to identify the
reasons of such risky ecosystem. By analyzing recorded data on river pollution from 2007-
2012, we found that river pollution has high correlation with the gross domestic products, not at
the same year, but for two years prior that indicates a lag time. We also found that under-five
child mortality has strong correlation with the river pollutions. The development-pollutionhealth
triangular cycle needs to be put in a balanced to ensure the nation prosperity and
sustainability of the nation.
Chemical toxins and organic contaminants such as hydrocarbons and dyes are major global
contaminants with countless tones of those chemicals are created yearly with a significant
amount release to the environment. In this work we screen the ability of a molybdenum-reducing
bacterium isolated from contaminated soil to decolorize various azo and triphenyl methane dyes
independent of molybdenum reduction. Biochemical analysis resulted in a tentative identification
of the bacterium as Enterobacter sp. strain Zeid-6. The bacterium was able to decolorize the azo
dye Orange G. The bacterium reduces molybdate to Mo-blue optimally at pH between 5.5 and
8.0 and temperatures of between 30 and 37 oC. Other requirements include a phosphate
concentration of 5 mM and a molybdate concentration of 20 mM. The absorption spectrum of the
Mo-blue produced was similar to previous Mo-reducing bacterium, and closely resembles a
reduced phosphomolybdate. Molybdenum reduction was inhibited by copper, lead, mercury and
silver which showed 36.8, 16.9, 64.9 and 67.6% inhibition to Mo-reducing activity of
Enterobacter sp. strain Zeid-6, respectively. The resultant molybdenum blue spectrum closely
resembles the spectrum of molybdenum blue from the phosphate determination method. The
ability of this bacterium to detoxify molybdenum and decolorize azo dye makes this bacterium
an important tool for bioremediation.
The pollution of heavy metals and toxic xenobiotics has become a central issue worldwide.
Bioremediation of these toxicants are being constantly carried out using novel microbes.
Molybdenum reduction to molybdenum blue is a detoxification process and mathematical
modelling of the reduction process can reveal important parameters such as specific reduction
rate, theoretical maximum reduction and whether reduction at high molybdenum concentration
affected the lag period of reduction. The used of linearization method through the use of natural
logarithm transformation, although popular, is inaccurate and can only give an approximate
value for the sole parameter measured; the specific growth rate. In this work, a variety of
models for such as logistic, Gompertz, Richards, Schnute, Baranyi-Roberts, Von Bertalanffy,
Buchanan three-phase and more recently Huang were utilized for the first time to obtain values
for the above parameters or constants. The modified Gompertz model was the best model in
modelling the Mo-blue production curve from Serratia marcescens strain DR.Y10 based on
statistical tests such as root-mean-square error (RMSE), adjusted coefficient of determination
(R2), bias factor (BF), accuracy factor (AF) and corrected AICc (Akaike Information Criterion).
Parameters obtained from the fitting exercise were maximum Mo-blue production rate (μm), lag
time (l) and maximal Mo-blue production (Ymax) of X (h-1), Y (h) and Z (nmole Mo-blue),
respectively. The application of primary population growth models in modelling the Moblue
production rate from this bacterium has become a successful undertaking. The model
may also be used in other heavy metals detoxification processes. The parameters
constants extracted from this work will be a substantial help for the future development
of further secondary models.
Observations on the effects of copper on the liver proteome of Puntius javanicus based on the
one dimensional PAGE was carried out. The liver was dissected from each fish, which was
separately treated with different concentrations of copper sulfate ranging from 0.1 to 5.0 mg/L.
The livers were extracted and one dimensional PAGE was performed under nonreducing
(native) and reducing (SDS)-PAGE. Several bands were resolved in the native PAGE with
probable candidates for the effect of copper observed showing an increased in the expression
and downregulation strongly associated with increasing copper concentrations. This study
showed that high concentrations of copper significantly alters P. javanicus liver at the proteome
level, and preliminary screening based on one dimensional PAGE is considered rapid and
simple to assess the toxicity effect of copper before more advanced and extensive assesment
with a second dimensional PAGE is carried out.
Contamination of organic xenobiotic pollutants and heavy metals in a contaminated site allows
the use of multiple bacterial degraders or bacteria with the ability to detoxify numerous toxicants
at the same time. A previously isolated SDS- degrading bacterium, Acinetobacter baumannii
strain Serdang 1 was shown to reduce molybdenum to molybdenum-blue. The bacterium works
optimally at pH 6.5, the temperature range between 25 and 34°C with glucose serves as the best
electron donor for molybdate reduction. This bacterium required additional concentration of
phosphate at 5.0 mM and molybdate between 15 and 25 mM. The absorption spectrum of the
molybdenum blue obtained is similar to the molybdenum blue from other earlier reported
molybdate reducing bacteria, as it resembles a reduced phosphomolybdate closely. Ag(i), As(v),
Pb(ii) and Cu(ii) inhibited molybdenum reduction by 57.3, 36.8, 27.7 and 10.9%, respectively, at
1 p.p.m. Acrylamide was efficiently shown to support molybdenum reduction at a lower
efficiency than glucose. Phenol, acrylamide and propionamide could support the growth of this
bacterium independently of molybdenum reduction. This bacterium capability to detoxify several
toxicants is an important tool for bioremediation in the tropical region.
Molybdenum, an emerging pollutant, has being demonstrated recently to be toxic to
spermatogenesis in several animal model systems. Metal mines especially gold mine often use
cyanide and hence isolation of metal-reducing and cyanide-degrading bacteria can be useful for
the bioremediation of these pollutants. Preliminary screening shows that three cyanide-degrading
bacteria were able to reduce molybdenum to molybdenum blue (Mo-blue) when grown on a
molybdate low phosphate minimal salts media. Phylogenetic analyses of the 16S rRNA gene of
the best reducer indicates that it belongs to the Serratia genus. A variety of mathematical models
such as logistic, Gompertz, Richards, Schnute, Baranyi-Roberts, von Bertalanffy, Buchanan
three-phase and Huang were used to model molybdenum reduction, and the best model based on
statistical analysis was modified Gompertz with lowest values for RMSE and AICc, highest
adjusted R2 values, with Bias Factor and Accuracy Factor nearest to unity (1.0). The reduction
constants obtained from the model will be used to carry out secondary modelling to study the
effect of various parameters such as substrate, pH and temperature to molybdenum reduction.
The 3D structure of the insecticidal protein Cry1Ba4 produced by B. thuringiensis subsp.
Entomocidus HD-9 was determined using homology modelling. From the model built, we have
been able to identify the possible sites for structure modification by site-directed mutagenesis.
The mutation was introduced at the conserved region of -helix 7 by substituting the
hydrophobic motif that comprises alanine 216, leucine 217 and phenylalanine 218 with arginine.
Wild and mutant Cry1Ba4 genes were cloned into pET200/D-TOPO and expressed in the
expression host. The result suggests that mutant Cry1Ba4 protein was less toxic to the larvae
Plutella xylostella compared to the wild-type. In conclusion, alteration in the structure of
Domain I had left an impact on the toxicity of Cry1Ba4 against P. xylostella.
The volume of contaminated rivers in Malaysia continues to keep rising through the years. The
cost of instrumental monitoring is uneconomical and prohibits schedule monitoring of
contaminants particularly heavy metals. In this work, a rapid enzyme assay utilizing the
molybdenum-reducing enzyme as an inhibitive assay, prepared in crude form from the
molybdenum-reducing bacterium Serratia sp. strain DRY5 has been developed for monitoring
the heavy metals mercury, silver, copper and chromium in contaminated waters in the Juru
Industrial Estate. The crude enzyme extract transformed soluble molybdenum
(phosphomolybdate) into a deep blue solution, which is inhibited by heavy metals such as
mercury, silver, copper and chromium. The IC50 and Limits of Detection (LOD) values for
mercury, copper, silver and cadmium were 0.245, 0.298, 0.367, 0.326, and 0.124, 0.086, 0.088
and 0.094 mg L-1, respectively. The assay is rapid, and can be carried out in less than 10 minutes.
In addition, the assay can be carried out at ambient temperature. The IC50 values for these heavy
metals are more sensitive than several established assays. Water samples from various locations
in the month of November from the Juru Industrial Estate (Penang) were tested for the presence
of heavy metals using the developed assay. Enzyme activity was nearly inhibited for water
samples from several locations. The presence of heavy metals was confirmed instrumentally
using Atomic Emission Spectrometry and a Flow Injection Mercury System. The assay is rapid
and simple and can be used as a first screening method for large scale monitoring of heavy
metals.
Mass production of fish broodstock with high quality eggs requires the knowledge on the chemical composition and physiochemical properties of vitellogenin (Vtg) during ovulation. Vtg is an egg yolk precursor phospholipoglycoprotein, and has been analysed to evaluate the reproductive conditions and determine the spawning period in captive and wild fish. In this study, Vtg was induced in male H. nemurus through three intramuscular injections of 17-estradiol (E2). The Vtg was purified from the serum using gel filtration chromatography and the purified protein was reduced via SDS-PAGE. One major polypeptide corresponding to 130 kDa was observed. Vtg identification was done using peptide mass fingerprint (PMF) from the trypsin digestion of male H. nemurus Vtg induced with E2. The sequence homology of H. nemurus AYLAGAAADVLEVGVR matched the Vtg of other fish species when analysed using MALDI-TOF. Vtg was confirmed by MASCOT at 95% significant level. The potential protein that controls the reproductive process and oocyte development isolated from this study was discussed to understand the structure and function of Vtg.
In this work, a temporal monitoring work for heavy metals from an effluent discharge point in
the Juru Industrial Estate was carried out using the protease extracted from garlic (Allium
sativum) as the principal bioassay system. casein-Coomassie-dye binding assay method has
utilized this purpose. The periodic sampling results for one day of a location in the Juru
Industrial Estate showed temporal variation of copper concentration coinciding with garlic
protease inhibition with the highest concentrations of copper occurring between 12.00 and 16.00
hours of between 3 and 3.5 mg/L copper. The crude proteases extracted from Allium sativum
successfully detect temporal variation of copper form this location. In conclusion, this assay
method has the potential to be a rapid, sensitive, and economic inhibitive assay for the largescale
biomonitoring works for the heavy metal copper from this area.
Investigation on in vivo effects of copper (Cu) on the ultrastructure of P. javanicus liver was
carried out using transmission electron microscopy (TEM). The addition of sublethal
concentration of 5 mg/L of Cu caused abnormalities on the bile canaliculi (BC) including
dilation and elongation compared to control and at lower concentrations of copper with a normal
round shape form. Findings from this study support an alternative histological assessment of the
effects of Cu concentration on P. javanicus liver.
In this work we assess the potential of acetylcholinesterase (AChE) from Oreochromis
mossambicus (Toman) as a sensitive test for the presence of insecticides. The partial purification
and characterization of a soluble AChE from Oreochromis mossambicus brain tissues using
affinity chromatography gel (procainamide–Sephacryl S-1000) showed that the partially purified
AChE was most active on acetylthiocholine (ATC) but had low activities on
propionylthiocholine (PTC) and butyrylthiocholine (BTC), indicating that the partially purified
fraction was predominantly AChE. Soluble AChE was partially purified 9.27-fold with a 91.12%
yield. The partially purified AChE displayed the highest activity on ATC at pH 7 and at 30oC
using 0.1 M Tris buffer. The enzyme exhibited Michaelis-Menten kinetic constants, Km, for
ATC, BTC and PTC at 36, 77 and 250 μM, respectively, and the maximum velocities, Vmax, were
18.75, 0.12 and 0.05 μmol/min/mg protein, respectively. Moreover, the AChE from
Oreochromis mossambicus presented comparable sensitivity to carbamates and
organophosphates insecticides than that from Electrophorus electricus and many other fish
AChE by comparing half maximal inhibitory concentration values. Therefore, the enzyme is a
valuable source for insecticides detection in Malaysian waters at lower cost.
Isolate JR1 was isolated from the polluted textile industry activities site in the Juru Penang area.
This bacterium was characterized as a gram-positive Bacillus bacterium and also gave a
positive biochemical test for catalase test and oxidase test. The isolate JR1 gave a maximum
decolourization of Amaranth dye under static conditions with the rate of decolorization of
98.82%. Seven variables which are pH, temperature (°C), ammonium acetate (g/L), glucose
(g/L), sodium chloride (g/L), yeast (g/L) and dye concentration (ppm) was run by using
Plackett-Burman design for the effective parameter of the decolourization of Amaranth. From
the seven variables, three effective variables which were ammonium acetate, glucose, and dye
concentration were further optimized by using a central composite design. The optimum value
of ammonium acetate concentration at 0.74 g/L, glucose concentration at 3.0 g/L and a dye
concentration at 58.1 ppm gave the highest percentage of decolourization. Thus, this isolate
could provide an alternate solution in removing toxic dyes from environments.
The Q10 value is tied to an increase in the surrounding temperature with an increase in 10 ◦C,
and usually resulted in a doubling of the reaction rate. When this happens, the Q10 value for the
reaction is 2. This value holds true to numerous biological reactions. To date, the Q10 value for
the biodegradation of phenol is almost not reported. The Q10 values can be determined from the
Arrhenius plots. In this study, the growth rate or biodegradation rates in logarithmic value for
the bacterium Pseudomonas sp. AQ5-04 was plotted against 1000/temperature (Kelvin) and the
slope of the Arrhenius curve is the value of the Ea, which was utilized to obtain the Q10. The
value obtained in this work was 1.834, which is slightly lower than the normal range of between
2 and 3 for the biodegradation rates of hydrocarbon in general and shows that this bacterium is a
very efficient phenol-degrading bacterium.
Environmental pollution is one of the major concerns in the 21st century; where billions of tonnes
of harmful chemicals are produced by industries such as petroleum, paints, food, rubber, and
plastic. Phenol and its derivatives infiltrate the ecosystems and have become one of the top major
pollutants worldwide. This review covers the major aspects of immobilization of phenoldegrading
bacteria as a method to improve phenol bioremediation. The use of various forms of
immobilization matrices is discussed along with the advantages and disadvantages of each of the
immobilization matrices especially when environmental usage is warranted. To be used as a
bioremediation tool, the immobilized system must not only be effective, but the matrices must be
non-toxic, non-polluting and if possible non-biodegradable. The mechanical, biological and
chemical stability of the system is paramount for long-term activity as well as price is an
important factor when the very large scale is a concern. The system must also be able to tolerate
high concentration of other toxicants especially heavy metals that form as co-contaminants, and
most immobilized systems are geared towards this last aspect as immobilization provides
protection from other contaminants.