Molybdenum is an emerging pollutant. Bioremediation of this heavy metal is possible by the
mediation of Mo-reducing bacteria. These bacteria contain the Mo-reducing enzymes that can
conver toxic soluble molybdenum into molybdenum blue; a less soluble and less toxic form of the
metal. To date only the enzyme has been purified from only one bacterium. The aim of this study is
to purify the Mo-reducing enzyme from a previously isolated Mo-reducing bacterium Bacillus
pumilus strain Lbna using ammonium sulphate fractionation followed by ion exchange and then
gel filtration. Two clear bands were obtained after the gel filtration step with molecular weights
of 70 and 100 kDa. This indicates that further additional purification methods need to be used
to get a purified fraction. Hence, additional steps of chromatography such as hydroxyapatite or
chromatofocusing techniques can be applied in the future.
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.
Phenolic compounds or phenols are a group of aromatic compounds that comprises a hydroxyl
group (OH) that is directly bonded to an aromatic ring. Phenols are injurious to organisms even
at even low concentrations with many of them are categorized as dangerous pollutants because of
their likely harm to human well-being. This review attempts to discuss the various merits and
demerits of immobilization matrices employed for phenol-degrading microorganisms’
immobilization. One of several key points of cellular immobilization is the capacity to protect
bioremediation agents towards toxic levels of specific toxicants and safeguarding from predatory
microorganisms. However, this shielding course of action should never impede the diffusion of
substrates into the pores of the immobilization structure. In the end the choice of a particular
immobilization method will strongly hinge on aspects of economy, safety and efficacy.
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.
Acetylcholinesterase (AChE) is usually used as an inhibitive assay for insecticides. A lesserknown
property of AChE is its inhibition by heavy metals. In this work, we evaluate an AChE
from brains of Clarias batrachus (catfish) exposed to wastes from aquaculture industry as an
inhibitive assay for heavy metals. We discovered that the AChE was inhibited completely by
Hg2+, Ag2+, Pb2+, Cu2+, Cd2+, Cr6+ and Zn2+ during initial screening. When tested at various
concentrations, the heavy metals exhibited exponential decay type inhibition curves. The
calculated IC50 (mg/L) for the heavy metals Ag2+, Cu2+, Hg2+, Cr6+ and Cd2+ were 0.088, 0.078,
0.071, 0.87 and 0.913, respectively. The IC50 for these heavy metals are comparable, and some
are lower than the IC50 values from the cholinesterases from previously studied fish. The assay
can be carried out in less than 30 minutes at ambient temperature.
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.
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.
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.
Acrylamide is a synthetic monomer that has been classified as toxic and carcinogenic apart
from its diverse application in the industry. Its application is in the formation of
polyacrylamide. Polyacrylamide usage is diverse and is found as herbicide formulation, as soil
treatment agent and in water treatment plants. Deaths and sickness due to the accidental
exposure to acrylamide have been reported while chronic toxicity is also a source of the
problem. This review highlighted the toxic effect of acrylamide to various organisms like
human, animal and plant. This review also discusses on the potential use of biological
technologies to remediate acrylamide pollution in the environment and the degradation
pathways these microorganisms utilize to assimilate acrylamide as a nitrogen, carbon or both as
carbon and nitrogen sources.
Heavy metals pollution has become a great threat to the world. Since instrumental methods are
expensive and need skilled technician, a simple and fast method is needed to determine the
presence of heavy metals in the environment. In this work, a preliminary study was carried out
on the applicability of various local plants as a source of protease for the future development of
the inhibitive enzyme assay for heavy-metals. The crude proteases preparation was assayed using
casein as a substrate in conjunction with the Coomassie dye-binding assay. The crude protease
from the kesinai plant was found to be the most potent plant protease. The crude enzyme
exhibited broad temperature and pH ranges for activity and will be developed in the future as a
potential inhibitive assay for heavy metals.
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.
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.
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.
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.
In this study, a novel glyphosate-degrading shows the ability to reduce molybdenum to
molybdenum blue. The enzyme from this bacterium was partially purified and partially
characterized to ascertain whether the Mo-reducing enzyme from this bacterium shows better or
lower efficiency in reducing molybdenum compared to other Mo-reducing bacterium that only
exhibits a single biotransformation activity. The enzyme was partially purified using ammonium
sulphate fractionation. The Vmax for the electron donating substrate or NADH was at 1.905 nmole
Mo blue/min while the Km was 6.146 mM. The regression coefficient was 0.98. Comparative
assessment with the previously characterized Mo-reducing enzyme from various bacteria showed
that the Mo-reducing enzyme from Burkholderia vietnamiensis strain AQ5-12 showed a lower
enzyme activity.
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.
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.
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.
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.