Laccases are multidomain copper containing proteins acting on phenolic and non-phenolic compounds. They are industrially relevant enzymes performing diverse oxidative functions including dye decolorization, food processing, organic synthesis and bio-remediation. Laccases exist in all domains of life. Laccases from fungal species are well characterized for industrial utilization. However, Laccases from bacterial genera are comparatively less characterized. In comparison with fungal laccases bacterial laccases are heat stable and halide tolerant, the properties desired for industrial applications. The present study describes the identification, cloning, gene expression and characterization of laccases from two sources (i) from thermophilic Geobacillus thermopakistaniensis and (ii) from mesophilic Bacillus subtilis strain R5. Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (GenBank accession number ESU71923) annotated as laccase, (named as Gt-Lac). Gt-Lac gene was having 825 nucleotides, encoding a protein of 273 amino acids. The BLAST search showed that Gt-Lac does not display sequence similarity with characterized laccases of Bacillus subtilis, Streptomyces coelicolor and Thermus thermophilus. Gt-Lac shared highest homology with laccases of a new protein family, DUF152, like Kurthia huakuii (32%), RL5 laccase from bovine rumen metagenome (31%), and Thermobifida fusca (28%). To examine the properties of ESU71923, Gt-Lac was cloned in expression vector (pET-21a) and mobilized to E. coli for the production of recombinant enzyme. However, the purified recombinant protein did not exhibit any laccase-like activity even when produced in the presence of copper ions. Expression of Gt-Lac gene at low temperatures and in the presence of zinc also failed to produce an active enzyme. Atomic absorption spectroscopy could detect only zinc atom instead of four coppers that most laccases possess. Based on these results it was suggested that ESU71923 does not encode a functional laccase. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 (named as Gt-Cuo) was prepared in Escherichia coli. Gt-Cuo gene encoded a protein of 503 amino acids with a molecular weight of about 60 kDa. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7–7.5. Gt-Cuo was copper dependent and a five-fold increase in laccase activity was observed in the presence of 100 μM copper sulfate. When using halide donors, a 7-fold and 5-fold increase in laccase activity was observed with 500 mM NaBr and NaCl, respectively. Gt-Cuo showed good stability towards organic solvents. Moreover Gt-Cuo was able to decolorize several synthetic dyes with highest rate of color removal observed for indigo caramine. In conclusion, this is the first report about the identification of gene, from genus Geobacillus, responsible for true laccase activity having potential to be used for biotechnological applications. The third gene of the study, (named as Bsu-Lac) laccase from Bacillus subtilis was composed of 513 amino acids with calculated molecular weight of 58498.99 Da. When expressed in E. coli the recombinant Bsu-Lac was produced as inclusion bodies which were tried to refold by denaturing in urea but the refolded sample was inactive. Co-expression of Bsu-Lac gene with a chaperonin gene also failed to solubilize the inclusion bodies. Finally expression of Bsu-Lac gene was taken in pET-28a(+) at low temperature and the protein was purified by nickel affinity chromatography. The enzyme was found to function optimally at 55 °C and a pH of 7. The laccase activity of Bsu-Lac was dependant on copper only during protein production rather than adding in assay mixture, showing the importance of copper for proper protein folding. The enzyme was stable in 10% of all tested organic solvents. The Bsu-Lac activity was stable at 80 °C for 150 min. When the protein was incubated with various concentrations of urea, structural stability was found even at 8 M urea. The recombinant protein was able to degrade various synthetic dyes with the highest rate of dye degradation for orange G, thus having potential for various industrial applications.
The Immensely Merciful to all, The Infinitely Compassionate to everyone.
104:01 a. Woe to every slanderer and backbiter,
104:02 a. as also the one who amasses wealth without spending a part of it on charity, and keeps adding and counting it over and over again,
104:03 a. thinking - in his ignorance - that his wealth will make him live forever.
104:04 a. Never! b. It is not like what he thinks. c. On the contrary, he will certainly be flung into the Crushing Fire - that which breaks its inmates to pieces.
104:05 a. And what may enable you to comprehend the Crushing Fire?
104:06 a. The Crushing Fire is Allah’s Fire, fiercely set ablaze,
104:07 a. which will leap up and penetrate deep into sinful peoples’ hearts and shall burn within as without.
104:08 a. It will definitely be closing upon them from all directions,
104:09 a. in towering and extending columns of flames.
Due to the requirement of Muslim Ummah in current issues a few rules were added in Islamic Jurisprudence. Among these new added rules is Al-Istehsan. The article discussed this rule covering the points given below: - Various definitions ofAl-Istehsan and its explanation. - Five types; Istehsan by nuss, Istehsan by ijmaa, Istehsan by urf, Istehsan by need, istehsan by qiyas. - Authenticity of Al-Istehsan by citing different opinions of the scholars. - Difference between Al-Istehsan and qiyas
Diabetes Mellitus (DM) is an advanced and chronic endocrine disorder characterized by insufficiency of insulin secretion from pancreatic beta cells or of insulin resistance in peripheral tissues such as liver, adipose tissues and skeletal muscles. Glucokinase (ATP: D-glucose-6-phosphotransferase; GCK), initiates glycolysis and acts as a glucose sensor and metabolic signal producer in liver and pancreas. The present study investigated differences in allelic frequencies between 60 individual diabetics with typeII and 60 healthy controls. Clinical manifestations identified, GCK gene was selected analyzed data were compared to RFLP of the glucokinase gene and results confirmed about mutation. There were also significant differences (P<0.0001) in glycation levels (0.90, 0.4838mole/mole), random blood sugar (348.8, 105.8 mg/dl), cholesterol levels (235.3, 161.8 mg/dl), low density lipoprotein in diabetic subjects (155.3, 28.46 mg/dl) and in healthy donors. GCK gene mutations were found in 70% of the patients while 30% are non-mutated. The basic aim of this study is to understand the mechanism and genes which are responsible for the prevalence of diabetes and to investigate new trends and methods used for the treatment of this disease. A hereditary variation of a gene is mentioned in this study, it is a change in a nucleotide sequence with an addition, deletion, or investment of a nucleotide sequence. In particular, genetic variation in a gene includes a single nucleotide polymorphism. Samples show the mutation insertion, deletion and insertion/deletion. PCR-sequencing showed qualitative differences in diabetic patients in comparison to non-diabetics. Glucokinase is the most important component in glucose detection of pancreatic islet beta cells in diabetes because glucokinase mutations can be one of the most common single gene disorders described. It is known that a genetic variation of a human glucokinase gene, including a point mutation, causes MODY, concentration of plasma glucose increased and it is supposed to be the cause of diabetes of the present study subjects. The main aspects of the work were designed to investigate immunological cross-reactivity between viral antigens and pancreas-specific self-antigens. This is to investigate, hypothesis that type1 diabetes may be triggered following infection with commonly occurring viruses such as coxsackie B, rotavirus, rubella, mumps and cytomegalovirus. The ANOVA of peripheral blood CD3+ T cells in healthy controls expresses higher level (1.475 ± 0.2930), than the diabetic patients (0.685±0.0927). CD14+ T-cell on monocyte1 frequency were analysed by flow-cytometry in the same Diabetes Mellitus patients expressing the higher level in healthy control (10.79±9.225) when compared to diabetic subjects (2.615±0.5850) and CD14+ on monocyte2 in healthy subjects’ level is higher (11.41±9.925) as compared to the diabetes (2.415±1.575). B-Cell marker CD19+ in healthy donors show higher significant level (5.085±5.055) and in diabetic subjects it is comparatively lowers (3.615±1.825). In conclusion, this study proved that oxidative stress increases the production of ROS causes the hyperglycaemic control stimulates the failure of different cells like lymphocytes subsets ultimate causes the cell death.