Papers by Dr. Md. Asaduzzaman
Parasporin, another type of δendotoxin from Bacillus thuringiensis (Bt), renowned for its highly ... more Parasporin, another type of δendotoxin from Bacillus thuringiensis (Bt), renowned for its highly specific and preferential toxicity against different cancer cells, could be a potentially safe anticancer therapeutic. Considering the current cancer situation, isolation and molecular characterization of parasporin-producing Bt isolates were aimed. Hence, Bt isolates were obtained through a series of tests viz. heat treatment, acetate selection, starch hydrolysis, lecithinase activity, and microscopy for parasporal proteins. Then the biochemical properties, plasmid, parasporin gene, and δendotoxin protein profiles were checked. Primers were designed for ps gene detection and the molecular identification of the isolates was performed by 16S rRNA gene sequence analysis. From the tested samples (n=128), 91 Bt isolates were obtained and of them, 28 non-hemolytic Bt isolates were selected as potential parasporin producers. Plasmid profile analysis of these isolates revealed that nonhemolytic...
Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent ... more Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent for tomato gray mold. The genome of B. subtilis strain BAB-1 was fully sequenced and annotated, genes encoding the antifungal active compound were identified, and multiple sets of regulatory systems were found in the genome.
The scarcity of hygienic drinking water is a normal phenomenon in the coastal areas of Bangladesh... more The scarcity of hygienic drinking water is a normal phenomenon in the coastal areas of Bangladesh due to the high salinity of ground water. The inhabitants of this locality, therefore, live on alternative supplies of water including rain-fed pond water, and rain-water with persistent complex microbial interactions therein, often contaminated with life-threatening pathogens. Hence, this study was aimed at analyzing the prevalence of Vibrio cholerae (Vc) in the alternative drinking waters of Mathbaria, a coastal subdis-trict neighboring the Bay of Bengal, the efficacy of pond sand filter (PSF) and the co-association among Bacillus-like spore formers (Sf) and Vc. Vc presumably entrapped into the membrane filter was enriched in alkaline peptone water medium and was isolated on selective thiosulfate-citrate-bile salts-sucrose and taurocholate-tellurite-gelatin agar media. They were finally identified by immunochromatographic one step rapid test and serology test. A total of 26% Vc positive samples were obtained out of 100 [ponds—48, household (HH)—29, and PSFs—23] where 13% cases were pathogenic (Vc O1) and 13% were non-pathogenic (Vc non-O1/non-O139). The distribution of Vc as observed was 33, 26, and 13.8% in waters derived from pond surface, PSF, and HH reservoirs, respectively, and for pathogenic type, it was 62.5%, 50%, and nil, respectively. Although none of the samples was identified with pathogenic Vc O139, the statistics represents a significant and augmentative risk of cholera outbreak in the focused area. The antibiotic sensitivity pattern in this study resembled the trend observed during last few years for Vc. The PSF demonstrated its inability to remove Vc from any of the samples and in addition, the filter itself was evidenced to be the source of pathogens and spores in further contamination and transmission. The development of biofilm in the PSF could be hypothesized as the reservoir in contaminating pathogen-free water samples. From the test of homogeneity, the risk levels of alternative water sources were estimated equal regarding Vc. Simultaneously, it was determined statistically that the prevalence of Vc, by no means, is influenced by Bacillus-like Sf be it for pond surface, HH, or PSF derived water.
Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marce... more Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low K m 0.3 and 0.5 mg ml À1 and high V max 333 and 500 mmole min À1 at 40 °C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (E a) and temperature quotient (Q 10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.
Aims: Bacillus licheniformis MZK-05 is a keratinolytic bacterium having potential in dehairing of... more Aims: Bacillus licheniformis MZK-05 is a keratinolytic bacterium having potential in dehairing of leather and feather hydrolysis. The present study aimed at to improving the production level of keratinase through gene cloning and expression of recombinant keratinase. Methodology and results: Bacillus licheniformis MZK-05 produced an amplicon of 1,156 bp in a polymerase chain reaction while targeting the gene, kerA, responsible for the enzyme keratinase. The amplicon was subsequently cloned into the plasmid vector pGEX-6p-2 for expression in Escherichia coli BL21. A 58 kD GST-KerA fusion protein was expressed upon IPTG induction which was eventually cleaved by PreScission protease that produced a 39 kD protein. A corresponding increase in proteolytic (312 U/mL) and keratinolytic (196 U/mL) activity were observed with the expressed keratinase. Specific enzyme activities for protease and keratinase, an indication of efficiency of the enzyme, were 2621.84 U/mg and 1647 U/mg, respectively and the specific keratinase activity was the highest activity ever reported by any recombinant bacterial strain. Conclusion, significance and impact study: Since the production of keratinase by wild type strain is limited to a certain level, the industrial need could be met by improving the production level through gene cloning and expression of recombinant keratinase. In this connection, the cloning of kerA gene from B. licheniformis MZK-05 into pGEX-6p-2 vector, its expression in Escherichia coli BL21 host and prediction of 3-D model of the expressed protein were performed which will be the basis for industrial production of keratinase in Bangladesh.
Integrated Pest Management (IPM) and bio-intensive pest management (BIPM) (where Bt biopesticide ... more Integrated Pest Management (IPM) and bio-intensive pest management (BIPM) (where Bt biopesticide is an indispensible component of it) are the suggested alternatives of chemical pesticides. So, a holistic approach to the isolation and detection of potential Bt strains, production at industrial scale and administration in the field is necessary to include Bt biopesticide in the IPM and BIPM of Bangladesh. In this connection, the bioinsecticide prepared from potential indigenous Bt strain JSc1 was applied in cabbage, cauliflower and organic tea farming and was found to be efficient in controlling the target lepidopteran pests such as Helicoverpa armigera, Spodoptera litura, Plutella xylostella, Hyposidra spp. etc. Results indicated that more than 85% of the treated crops were protected from the infestation and destruction by the pests. Obtained data analyzed using ANOVA test suggested the inclusion of Bt biopesticide in the IPM of Bangladesh as no such differences were observed with the chemical pesticides currently in use.
The present study was carried out to develop a sustainable production mcdium using locally avlila... more The present study was carried out to develop a sustainable production mcdium using locally avlilabl€ cheap raw mat€rials for biopesticide production by Bacillus thuingiensrs subsP. *.r$tari (Bt*) HD-73. In submerged fermentat'on (SmF) condition, the cotrvetrtional Luria-Berta[i (LB) medium which was enriched with nitrogen source (107o d€frtted soybean meal) supported 28,579lo sporulation aa nd l2S"/" €ndotoxin incr€as€ over LB (atone). The effect of cystine on sporulation atrd eudotoxin synthesis was highly protroutrced in LB-soyb€an medium (LBS) with a ratrg€ of lg,54y" and 13t,357o higher endotoxin yield respectively in SmF conditioo' while basal salts supplemented in soybeatr-cystine (sMc) medium, it result€d in 7.659/o €ndotoxin yield compared to LB-soyb€an-cystin€ (LBSC) medium. Addition of molasses baltnced th€ c: N ratio io the sMc medium thus h€lping 84.859/. higher eodotoxin syuthesis after 24 hours lermentation. Substitutiou of basal safts with cost effective s€a water yi€lded abott 19.27"h less endotoxin. The optimum medium thus obtained consisting of soybean extyact-molasscs-cystine with s€a water was used in 3.{} L bioreactor cultivation for endotoxin synthesis by Btk HD-13 under 30%o saturation of dO2 through cascade of agitstion and aeration. The production rate obtained was 1.67 fold higher i|r bioreactor than in shake flask culture'
Genetic diversity is highly relevant and significant in discovering novel insecticidal genes in B... more Genetic diversity is highly relevant and significant in discovering novel insecticidal genes in Bacillus thuringiensis (Bt) strains and to deal with the problems of emerging insect resistance towards Bt biopesticides. In view of this, Random Amplified Polymorphic DNA (RAPD)-PCR analysis was performed with a decamer AGCTCAGCCA for molecular typing of 177 Bt strains of Bangladesh to determine their genetic diversity. These Bt strains were allocated into 15 genomic types with their binary matrices as determined from the dendrogram based on a standardized distance in scale bar. Genotype 9 and 11 were the largest among others, each containing more than 25% of the Bt strains. The average diversity index, as deduced for each group by cluster: isolate ratio at a specific distance, was higher for locations (0.27 ± 0.098) than that for biotypes (0.23 ± 0.046) which indicates an unmingled and vertical transfer of biochemical properties among the strains. Prevalence of agriculturally important subgroups of cry1 gene in indigenous Bt strains was also determined where cry1Aa and cry1Ca gene were found to be the most prevalent (21.74%). While analyzing the distribution pattern of cry genes, they were observed to be present in all RAPD- genotypes but genotype 10 and were most prevalent in genotypes 1, 6 and 9. The phylogeny reconstruction among the strains was performed by neighbor-joining method with the 16S rRNA gene sequences and the correlation among the phylogeny, RAPD genotypes, Biotypes and presence of cry genes were analyzed.
Bacteriocins are ribosomally synthesized antibacterial peptides which have the potential to be us... more Bacteriocins are ribosomally synthesized antibacterial peptides which have the potential to be used as natural food
preservatives as well as alternative to antibiotics. Here, we report the production of bacteriocin-like inhibitory
substances (BLIS) from the indigenous strains of Bacillus thuringiensis. Deferred antagonism bacteriocin assay and
agar well diffusion methods suggested that several of the tested strains have high levels of bacteriocin-like activity
against the common food-borne pathogens, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus.
These bacteriocins are generally produced at the mid-logarithmic phase of growth with optimum temperature of 37
ºC, pH-7.0 and 24 h of incubation. Heat stability assay demonstrated that the bacteriocins produced from the strains
are highly heat stable and can retain activity up to 100 ºC. Our study suggests that these bacteriocins may be
potential candidates for use as biodegradable natural food preservatives and alternative antimicrobial agents to solve
the increasing trends of problems of antibiotic resistance.
Key words: Bacteriocin, Bacillus thuringiensis, antimicrobial activity, Bangladeshi strains, food-borne pathogens
Bactrocera cucurbitae (melon fruit fly) is one of the most detrimental vegetable-damaging pests i... more Bactrocera cucurbitae (melon fruit fly) is one of the most detrimental vegetable-damaging pests in Bangladesh. The toxicity of Bacillus thuringiensis (Bt) has been reported against a few genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring cry1A-type genes were, therefore, assayed in vivo against the 3rd instar larvae of B. cucurbitae in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8% and 11.16%, respectively, of the test strains (n=224) while their prevalence was greatest in biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-73 (96%) and the indigenous Bt JSc1 (93%). LC50 and LC99 values were determined to be 6.81 and 8.32 for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause of toxicity and its variation among the strains was found to be correlated with the synergistic toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus will help in developing efficient and eco-friendly control measures such as Bt biopesticides and transgenic Bt cucurbits.
Nevertheless, emphasis should be given to promote the industrial growth in Bangladesh for sustain... more Nevertheless, emphasis should be given to promote the industrial growth in Bangladesh for sustainable economic development while dependency on agricultural economy alone is at a stake due to urbanization, natural calamities (erosion, flood, salinity and droughts) and increase in people land ratio. The biotech industries based on microbial catalysis has got priority globally due to its unique potentialities over chemical process particularly in the bioprocess based on cheap agricultural raw materials, low energy involvement and rare environmental pollution. Considering the annual import of the bio-products, local market demand, availability of critical skilled man power and resources in Bangladesh, the biotech industries establishment should be believed favorable. In this connection, the regulating bodies should assess the advancement and potentialities of local existing Research & Development sectors and take essentials steps for achieving the goals. In view of the above, our research development and findings on bioprocess for industrial enzymes for leather processing and mass production of Bt biopesticde for eco-friendly pest management are described. The results obtained are ready to be promoted to commercial level which is definitely a stimulatory and pioneering interface for the contemporary and near future biotech industries in Bangladesh.
The present study was aimed at designing a cost effective medium for increasing the δ-endotoxin (... more The present study was aimed at designing a cost effective medium for increasing the δ-endotoxin (Cry protein) synthesis by Bacillus thuringiensis (Bt) kurstaki HD-73 and indigenous Bt JSc1 harboring potential cry genes active against Lepidoptera insect orders. In this regard, locally available cheap molasses as carbon source, soybean extract as nitrogen source, sea water as trace elements source, cystine as sporulation/growth factor were considered to design a cost effective medium. Molasses and soybean extract in place of glucose and peptone in glucose-peptone (GP) medium supported 78.85% increase in δ-endotoxin synthesis in shake flask culture. The effect of cystine on endotoxin synthesis was highly pronounced in two media with a range of 80.32 to 110% higher δ-endotoxin under comparable fermentation conditions. While, substituting basal salts with sea water, Btk HD-73 yielded satisfactory and comparable endotoxin (74.3% of yield with basal salts). It was detected that the rapid decrease of endotoxin synthesis in the culture after 24 h was due to the degradation by the endogenous protease, synthesized with the progress of fermentation. This degradation of the endotoxin was much better protected (1.23 mg/ml endotoxin versus 0.312 mg/ml) by adding 4% ammonium sulfate in the optimized medium. The medium thus formulated with molasses, soybean extract, ammonium sulfate, cystine and sea water was then used in 3.0 L bioreactor cultivation for endotoxin synthesis by both Btk HD-73 and Bt JSc1 under 30% saturation of dO2 through cascade control of agitation and aeration producing a higher yield of δ-endotoxin (2.1 and 2.63 mg/ml, respectively). The present results may successfully be used for large scale production of Bt biopesticide in Bangladesh.
Bacillus thuringiensis (Bt) biopesticides, a recognized eco-friendly pest control agent, can be u... more Bacillus thuringiensis (Bt) biopesticides, a recognized eco-friendly pest control agent, can be used to reduce many problems associated with indiscriminate use of chemical pesticides such as environmental pollutions, public health problems, emergence of resistance among pests in many developing countries etc. Bt strains were, therefore, isolated from different ecosystems of Bangladesh and characterized based on biochemical typing, 16S rRNA gene analysis, plasmid and cry genes profiles. Bt index was calculated 0.86 in this study and variations in abundance and distribution pattern of 16 different biotypes were demonstrated within 316 indigenous Bt strains which was compared to the other parts of the world. Bt indiana (17.8%), Bt kurstaki (16.7%), and Bt thuringiensis (12.7%) were found to be the most prevalent in Bangladesh among other biotypes. Hemolytic activity was variable among the biotypes and it was maximum for Bt biotype 10 (100%). Plasmids in the biotypes indiana, kurstaki, thuringiensis, and israelensis were observed to occupy a wider range than other biotypes. The screening for insecticidal genes viz. cry1, cry2, cry3, cry4A, cry8, cry9, cry10, and cry11 in the native Bt strains revealed their presence in varied proportion rendering cry1, cry2, and cry3 the most abundant. The abundance of Bt strains, their diversities and the cry genes profile were thus analyzed in this study which will be the basis for further research development with Bt biopesticide in Bangladesh.
The indiscriminate use of agricultural pesticides has created serious health and environmental pr... more The indiscriminate use of agricultural pesticides has created serious health and environmental problems in many developing countries including Bangladesh. On the other hand, Bacillus thuringiensis kurstaki (Btk) has long been used commercially as pest control agents particularly against lepidopteran vegetable pests. In this study, new potential Btk-like isolates were collected from different sources (viz. soil, leaves, insects, stored product dust etc.) and regions of Bangladesh. Acetate selection-heat treatment-lecithinase production and haemolytic activity were initially performed to narrow down the spectrum for selection of Btk-likeisolates. Specific biochemical tests (Es+, Sa+, Le+, Su-) were performed to differentiate Btk from other subspecies. Based on these tests, of 148 Bt isolates, 27.7% (41 Bt isolates) were confirmed as Bt kurstaki isolates. The 41 isolates were investigated for the presence of cry1, cry1A and cry2A genes by PCR identification with gene specific primers and demonstrated some level of diversity with respect to major genes content in the strains. Nine of the isolates were found to carry all three genes while another nine contained both cry1 and cry2A genes. 29, 22 and 16 of the isolates contained singly cry1, cry2A or cry1A gene respectively. Plasmid DNA was purified which ranged from 12-15 kb in most of the isolates which were very similar to the plasmid pattern of Btk HD-73. Bioassay was performed with the isolates harbouring Lepidoptera specific cry1A genes against vegetable pest Bactrocera cucurbitae. Five of the isolates having higher activity close to Btk HD-73 were considered for determination of LC50 and LC90. The results will be a useful basis for developing effective bio-pesticides to decrease the use of hazardous chemicals in Bangladesh agriculture.
Bacillus thuringiensis (Bt) was investigated in three different habitats (vegetable and crops-cul... more Bacillus thuringiensis (Bt) was investigated in three different habitats (vegetable and crops-cultivated soils, phylloplanes and insect guts) of Bangladesh. A total of 61 Bacillus cereus-like isolates were obtained by selective methods and 57 of those were identified as Bacillus thuringiensis isolates based on their hemolytic activity, presence of parasporal crystal proteins, plasmid profile and crystal protein profile. The prevalence of Bt was highest (60%) in soil samples followed by leaf and insects. Five different types of parasporal crystal proteins (spherical, bipyramidal, irregular pointed, cuboidal and irregular shaped) were observed among the isolates which indicates the diversity of the local Bt isolates examined. In addition, confirmation of the strain identification was done using 16S rDNA gene sequencing. Plasmid analysis recovered from the isolates yielded at least one 15kb DNA, which is well comparable to the reference strain, B. thuriginesis kurstaki var. HD-73. Further similarity between the test and standard strains was observed while analyzing crystal proteins on a SDS-polyacrylamide gel that produced major bands of Cry1, Cry2 and Cry9 type proteins. Bioassay performed with isolates Bt-01i, Bt-25f and reference strain, Bt kurstaki var. HD-73 against pulse beetles Callosobrochus chinensis resulted LC 50 values of 0.30, 0.72 and 0.22 mg/ml crude proteins respectively which demonstrated their competency in Biopesticide production and application.
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Papers by Dr. Md. Asaduzzaman
preservatives as well as alternative to antibiotics. Here, we report the production of bacteriocin-like inhibitory
substances (BLIS) from the indigenous strains of Bacillus thuringiensis. Deferred antagonism bacteriocin assay and
agar well diffusion methods suggested that several of the tested strains have high levels of bacteriocin-like activity
against the common food-borne pathogens, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus.
These bacteriocins are generally produced at the mid-logarithmic phase of growth with optimum temperature of 37
ºC, pH-7.0 and 24 h of incubation. Heat stability assay demonstrated that the bacteriocins produced from the strains
are highly heat stable and can retain activity up to 100 ºC. Our study suggests that these bacteriocins may be
potential candidates for use as biodegradable natural food preservatives and alternative antimicrobial agents to solve
the increasing trends of problems of antibiotic resistance.
Key words: Bacteriocin, Bacillus thuringiensis, antimicrobial activity, Bangladeshi strains, food-borne pathogens
preservatives as well as alternative to antibiotics. Here, we report the production of bacteriocin-like inhibitory
substances (BLIS) from the indigenous strains of Bacillus thuringiensis. Deferred antagonism bacteriocin assay and
agar well diffusion methods suggested that several of the tested strains have high levels of bacteriocin-like activity
against the common food-borne pathogens, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus.
These bacteriocins are generally produced at the mid-logarithmic phase of growth with optimum temperature of 37
ºC, pH-7.0 and 24 h of incubation. Heat stability assay demonstrated that the bacteriocins produced from the strains
are highly heat stable and can retain activity up to 100 ºC. Our study suggests that these bacteriocins may be
potential candidates for use as biodegradable natural food preservatives and alternative antimicrobial agents to solve
the increasing trends of problems of antibiotic resistance.
Key words: Bacteriocin, Bacillus thuringiensis, antimicrobial activity, Bangladeshi strains, food-borne pathogens