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Application of Statistically Based Experimental Designs to Optimize Cellulase Production and Identification of Gene.

ABSTRACT: A natural bacterial strain identified as Bacillus amyloliquefaciens MBAA3 using 16S rDNA partial genome sequencing has been studied for optimization of cellulase production. Statistical screening of media components for production of cellulase by B. amyloliquefaciens MBAA3 was carried out by Plackett-Burman design. Plackett-Burman design showed CMC, MgSO4 and pH as significant components influencing the cellulase production from the media components screened by Plackett-Burman fractional factorial design. The optimum concentrations of these significant parameters were determined employing the response surface central composite design, involving three factors and five levels was adopted to acquire the best medium for the production of cellulase enzyme revealed concentration of CMC (1.84 g), MgSO4 (0.275 g), and pH (8.5) in media for highest enzyme production. Response surface counter plots revealed that middle level of MgSO4 and middle level of CMC, higher level of CMC and lower level of pH and higher level of MgSO4 with lower level of pH increase the production of cellulase. After optimization cellulase activity increased by 6.81 fold. Presence of cellulase gene in MBAA3 was conformed by the amplification of genomic DNA of MBAA3. A PCR product of cellulase gene of 1500 bp was successfully amplified. The amplified gene was conformed by sequencing the amplified product and sequence was deposited in the gene bank under the accession number KF929416. Response surface graph showing interaction effects between concentration of a CMC and MgSO4. b pH and CMC. c MgSO4 and pH.

SUBMITTER: Thakkar A

PROVIDER: S-EPMC4311583 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei. RESULTS:We have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes. CONCLUSIONS:Transcriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

Project description:BackgroundSystemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD) are manifestations of an autoinflammatory disorder with complex pathophysiology and significant morbidity, together also termed Still's disease. The objective of the current study is to set in silico models based on systems biology and investigate the optimal treat-to-target strategy for Still's disease as a proof-of-concept of the modeling approach.MethodsMolecular characteristics of Still's disease and data on biological inhibitors of interleukin (IL)-1 (anakinra, canakinumab), IL-6 (tocilizumab, sarilumab), and glucocorticoids as well as conventional disease-modifying anti-rheumatic drugs (DMARDs, methotrexate) were used to construct in silico mechanisms of action (MoA) models by means of Therapeutic Performance Mapping System (TPMS) technology. TPMS combines artificial neuronal networks, sampling-based methods, and artificial intelligence. Model outcomes were validated with published expression data from sJIA patients.ResultsBiologicals demonstrated more pathophysiology-directed efficiency than non-biological drugs. IL-1 blockade mainly acts on proteins implicated in the innate immune system, while IL-6 signaling blockade has a weaker effect on innate immunity and rather affects adaptive immune mechanisms. The MoA models showed that in the autoinflammatory/systemic phases of Still's disease, in which the innate immunity plays a pivotal role, the IL-1β-neutralizing antibody canakinumab is more efficient than the IL-6 receptor-inhibiting antibody tocilizumab. MoA models reproduced 67% of the information obtained from expression data.ConclusionsSystems biology-based modeling supported the preferred use of biologics as an immunomodulatory treatment strategy for Still's disease. Our results reinforce the role for IL-1 blockade on innate immunity regulation, which is critical in systemic autoinflammatory diseases. This further encourages early use on Still's disease IL-1 blockade to prevent the development of disease or drug-related complications. Further analysis at the clinical level will validate the findings and help determining the timeframe of the window of opportunity for canakinumab treatment.

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Application of Statistically Based Experimental Designs to Optimize Cellulase Production and Identification of Gene.

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