Isolation and identification of a new bacillus cereus strain and characterization of its neopullulanase

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of pullulanase applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantative analysis of the strain's pullulanase activity was carried out by the Dintrosalicyclic [DNS] acid-based assay. Thin layer chromatography [TLC] of the culture supernatant, identified the extracellular pullulanase as neopullulanase. Effects of temperature and pH on pullulanase activity were also studied. The optimum conditions for enzyme activity, as represented by 60°C and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30°C to 50°C. The effect of metal ions and reagents, such as Mg[+2], Ca[+2], Zn[+2], Cu[+2], Fe[+2], Ni[+2] on enzyme activity showed that Ca[+2] ions increased pullulan activity, whereas the other ions and reagents inhibited pullulanase activity. The ability of B. cereus SDK2 to produce high levels of neopullulanase stable at 60°C that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries

nested-splicing by overlap extension PCR improves specificity of this standard method

Background: splicing by overlap extension [SOE] PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function

Objectives: we introduced a nested-SOE-PCR [N -SOE-PCR] in order to increase the specificity and generating mutations in a gene by SOE-PCR

Materials and Methods: genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application

Results: in comparison to the conventional SOE-PCR, the improved method [i.e. N-SOE-PCR] increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products

Conclusions: by applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE

Isolation, cloning and sequence analysis of 1-aminocyclopropane-1-carboxylate deaminase gene from native Sinorhizobium meliloti

Many plant growth-promoting bacteria including Rhizobia contain the 1-aminocyclopropane-1-carboxylate [ACC] deaminase enzyme that can leave ACC, and thereby lower the level of ethylene in stressed plants. Drought and salinity are the most common environmental stress factors for plants in Iran. The main aim of this research was development of bio-fertilizers containing ACC deaminase enzyme which is very important in conditions of stressed drought and salinity. In this research 168 isolates of native Sinorhizobium meliloti were evaluated for ACC deaminase activity. These isolates were classified in four groups based on growth rate on ACC containing medium and enzyme activity. One isolate from each group was selected for molecular characterization. The nucleotide sequence of 16S rRNA gene of the selected isolates were determined. The ACC deaminase genes [acdS] on total and chromosomal DNA of S. meliloti KYA40, and KYA71 strains were isolated and cloned in pTZ57R/T vector and the obtained recombinant plasmids were used for sequence analysis. The sequence of acdS genes from strains KYA71 and KYA40 and corresponding proteins were analyzed with respect to available sequences in NCBI database. The 16S rRNA gene sequences of S. meliloti strains submitted to the GeneBank/NCBI database. The acdS gene of KYA71 may be located on chromosomal DNA and in KYA40 it is located on one of the mega plasmids. These two genes have 99% similarity with three nucleotide differences which only lead to a change in one amino acid 48, threonine in KYA40 acdS gene and methionine in KYA71. The comparison of amino acid sequences of KYA40 and KYA71 with other sequences in the database showed that the amino acids 37 to 58 in almost all strains were similar. Therefore, it was concluded that it was a conserved region in this location of acdS genes and any changes in this region may cause change in ACC deaminase activity

Cloning and expression of gumboro VP2 antigen in Aspergillus niger

Infectious Bursal Disease Virus [IBDV] causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger [A. niger]. Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG[-] protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. A number of pyrG[+] positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry

Expression enhancement in trastuzumab therapeutic monoclonal antibody production using genomic amplification with methotrexate

Trastuzumab [Herceptin] is a humanized monoclonal antibody [mAb] which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2[+] tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase [DHFR]. It is usually accepted that DHFR gene can be amplified in DHFR CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR

[Cloning virG gene and generation mutant construct of pGEM delta virG in order to induction recombination in native Shigella dysenteriae]

Shigellosis disease is the major causes of morbidity in children with diarrhea in Iran. The virG [icsA] gene plays a key role in pathogenesis and ability of invasion in shigella. The aim of this study was cloning, sequencing virG gene and developing a mutant construct pGEM delta virG in order to induction recombination in a native shigella for generation a live attenuated vaccine candidate strain. Initially, by use of biochemical tests, the native shigella strain was detected. The virG gene was cloned in pGEM-7zf vector and the nucleotide sequence was determined. According to the data of sequencing, digestion mapping of pGEMvirG vactor was obtained and a part of virG gene by using enzymatic digestion was removed. Finally, pGEM delta virG construct was transformed to E. coli by utilization of chemical transformation method. The native shigella strain by using biochemical tests was confirmed. The result of sequencing virG gene [native strain] was submitted in NCBI Genebank database. The pGEM delta virG construct contains a mutant construct of virG gene which 1751 bp was deleted through enzymatic digestion reaction and transformed in E. coli. Using the technique of allelic exchange based on the incident of recombination in bacteria is one of the most effective methods to develop a disruption in the target genes. This mutant construct can be applied in development of a live attenuated Shigella dysenteriae vaccine candidate.

Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences

Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene [btl2] was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors [pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA] were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies

Optimization of secretory expression of recombinant hGM-CSF in high cell density cultivation of recombinant Escherichia coli using Taguchi statistical method

Human granulocyte macrophage colony stimulating factor [hGM-CSF] has many therapeutic applications. In this study, in order to verify the purification process, the effect of carbon source, IPTG concentration and post-induction time on the secretion of recombinant hGM-CSF into the culture medium by recombinant Escherichia coli during high cell density cultivation were evaluated by using the Taguchi statistical method. The results indicated that glucose, 1mM IPTG and a time of 6 h post-induction, represented optimum conditions. The secreted hGM-CSF, overall volumetric productivity and purified hGM-CSF were 373 mg/l, 18 mg/l/h and 63 mg/l, respectively

In silico genome-wide screening for TnrA-regulated genes of Bacillus clausii

Bacillus clausii TnrA transcription factor is required for global nitrogen regulation. In order to obtain an overview of gene regulation by TnrA in B. clausii KSM-K16, the entire genome of B. clausii was screened for the consensus sequence, 5'-TGTNAN7TNACA-3' known as the TnrA box, and 13 transcription units were found containing a putative TnrA box. The TnrA targets identified in this study were tnrA, glnA, nrgA, nasFDEB, puc genes, licT, the two operons of the oligopeptide ABC transporter, lytR, transcriptional regulator of the Lrp/AsnC family, sodium-dependent transporter of SNF family, hyu genes and a biochemically uncharacterized protein

Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor

The mature core protein of the Hepatitis C virus [HCVC173] carrying pelB as a signal peptide [PelB::core] was overexpressed in Escherichia coli as 18% and 23.3% of the host's total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 +/- 1 mg HCVC173/g dry cell weight and an overall productivity of 51 +/- 1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant PelB::core protein was overexpressed as the inclusion body [IB] form, higher than the expected level when compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli

Cloning and enhanced expression of an extracellular alkaline protease from a soil isolate of Bacillus clausii in Bacillus subtilis

Alkaline proteases are of industrial importance, mainly in the detergent industry. In this study, the extracellular alkaline serine protease gene, aprE, from Bacillus clausii was amplified by PCR and further cloned and expressed in B. subtilis WB600 using the pWB980 expression vector. Protease activity of the recombinant B. subtilis WB600 harboring the plasmid pWB980/aprE reached up to 1020 U/ml, approximately 3-folds higher than the native B. clausii strain. Characterization of the recombinant alkaline protease by SDS-PAGE and zymogram analyses indicated a molecular weight of 31 kDa. DNA sequence analysis and the deduced amino acid sequence revealed 98% homology with the extracellular alkaline serine protease from B. clausii KSM-K16

[Functional screening of phosphatase-encoding genes from bacterial sources]

Phosphatase [APase] enzymes including phytases have broad applications in diagnostic kits, poultry feeds, biofertilizers and plant nutrition. Because of high levels of sequence diversity among phosphatases, an efficient functional screening method is a crucial requirement for the isolation of the encoding genes. This study reports a functional cloning screening method for the isolation of APase-encoding genes from bacterial genomic libraries in a medium containing a chromogenic substrate. The method was optimized to distinguish the desired signal from the background chromosomal APase activity. This screening method led to the isolation of two novel APase-encoding genes from Pseudomonas putida with no similarities to the known genes in the databases, indicating successful implementation of the developed method

Characterization of Pseudomonas aeruginosa in burn patients using PCR- restriction fragment length polymorphism and random amplified polymorphic DNA analysis

One of the major opportunistic pathogens in patients with burn injuries is Pseudomonas aeruginosa, which causes severe infections in burned patients. The objective of the study was to examine the molecular epidemiology of P. aeruginosa colonization in the burn unit of Shahid Motahari Hospital in Tehran, Iran. Restriction fragment length polymorphism [RFLP] and random amplified polymorphic DNA [RAPD] analysis were employed to study 127 clinical and two environmental P. aeruginosa isolates collected from January to June 2008. In RFLP, the PCR products of 16S rRNA gene were digested with restriction enzyme Alu I, Hae III, and Rsa I, and the fragments generated were analyzed by agarose electrophoresis. Molecular typing by RFLP did show no discriminatory power for P. aeruginosa isolates, but RAPD-PCR revealed eight different genotypes; RAPD 1 to RAPD8 in clinical and environmental isolates. RAPD1 was the major genotype in clinical [n=64, 50.4%] and environmental isolates [n=l, 50%]. The findings suggest that RAPD might have a superior typeabil-ity and discriminatory power over RFLP to study P. aeruginusa. Moreover, they highlight the need for further attention to the control of infection sources in Burn Units to prevent the transmission of the bacterium

Enhanced bioadsorption of cadmium and nickel by E. coli displaying a metal binding motif using CS3 fimbriae

Display of peptides on the surface of bacteria offers many new and exciting applications in biotechnology. Fimbriae is a good candidate for epitope display on the surface of bacteria. The potential of CS3 fimbriae of enterotoxigenic E. coli as a display system has been investigated. A novel cell surface display system with metal binding property was developed by using CS3 fimbriae. Short metal binding peptide, Gly-Cys- Gly-Cys-Pro- Cys- Gly- Cys- Gly as a cysteine rich peptide, was inserted into CS3 fimbriae and displayed on the surface of E. coli. Bacteria expressing hybrid pili with cysteine rich peptide could adsorb 392.5, 510 and 905 nmol of Ni2+, Cd2+ and Pb2+ per mg [dry weight] of cells, respectively, which are five-fold [nickel] and three-fold [cadmium] more than E. coli expressing native pili. Thus, expression of Cys-rich peptide enables bacteria to act as a metalloaffinity adsorbent. These results open the possibility for biosorption of heavy metal ions using engineered microorganisms