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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Antioxidant enzymes,Thermostability,Direct Mutagenesis,food supplements
- چکیده:
- چکیده انگلیسی: Background:Characterizing the structure and function of superoxide dismutase (SOD), as an antioxidant enzyme providing opportunities for its application in food supplements.
Objectives: In this study, the features of the Manganese-SOD of Lactococcus lactis (SDLL), subsp. cremoris MG1363, as probiotic bacteria, were determined on the basis of the computational methods.
Materials and Methods: The protein’s physicochemical properties and the prediction of its secondary structure were determined via the ProtParam server and the GOR program respectively. Moreover, the 3D structures of the proteins were constructed via the MODELLER on the basis of the homology method and the threading algorithm MUSTER. On the other hand, the structural stability of the models was assayed under the quasi-physiological conditions by the GROMACS program via the GROMOS96 43a1 force field in Linux system. Finally, using the Molecular Docking Studies, the functionality features of the models were predicted through their affinity with the corresponding substrates.
Results:The results revealed the physicochemical properties of the SDLL and a 3D model of a chain of the enzyme being similar to the SOD from the Bacillus Subtilis (SDBS). The model of the SDLL was checked for quality control purposes including the Ramachandran plot, the ERRAT and the Verifiy3D. The model was suggestive of the structural stability in quasi-physiological conditions; yet, less than that of the SDBS. Assessing the cause of the instability in the SDLL model was indicative of two unstable regions in the area far from the enzyme’s active position, they were considered suitable for mutagenesis. Accordingly, the loop substitution for the corresponding region of SDBS and the deletion of the loop positioned at the C terminal of SDLL resulted in a mutant of SDLL with more stability and appropriate affinity with the corresponding substrate.
Conclusion: In general, the study provides a new model of SDLL with certain thermostable features, and a new mutant with suitable stability and functionality on the basis of the direct mutagenesis being used in different applications.- انتشار مقاله: 12-02-1397
- نویسندگان: Nazanin Gholampour-Faroji,Monir-sadat Shakeri,Jafar Hemmat,Mohammad Rastegar-Moghadam,Aliakbar Haddad-Mashadrizeh
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Amylase,Industry,Thermal stability,Catalytic activity,Cel5E,Geobacillus stearothermophilus
- چکیده:
- چکیده انگلیسی: Background: Considering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable.
Objectives: Using in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function.
Materials and Methods: In this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity.
Results: A series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed.
Conclusions: Based on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.- انتشار مقاله: 15-04-1394
- نویسندگان: Ibrahim Torktaz,Jafar Hemmat,Ali Asghar Karkhane,Garshasb Rigi,Amin Rostami,Jafar Khezri,Reza Behroozi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Bacillus thermocatenulatus Lipase,capillary isoelectric focusing,Inclusion bodies,Periplasmic space,Tandem mass spectrometry
- چکیده:
- چکیده انگلیسی: 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.- انتشار مقاله: 10-07-1391
- نویسندگان: Ali Asghar Karkhane,Bagher Yakhchali,Ferdous Rastgar Jazii,Jafar Hemmat,Parvin Shariati,Mahvash Khodabandeh,Alireza Zomorodipoor
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Hepatitis C Virus,Recombinant protein,Overexpression,core protein
- چکیده:
- چکیده انگلیسی: 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.
- انتشار مقاله: 09-07-1390
- نویسندگان: Jafar Hemmat,Bagher Yakhchali,Khosro Khajeh,Ali Akbar Moosavi-Movahedi,Ali Asghar Karkhane
- مشاهده
- جایگاه : پژوهشی
- مجله: Advanced Research in Microbial Metabolites & Technology
- نوع مقاله: Journal Article
- کلمات کلیدی: Cellulase,Thermostability,Hot spring,Acidostability
- چکیده:
- چکیده انگلیسی: Cellulases with capability to have enzymatic functions at low pH and high temperature are important and include considerable portion of industrial enzymes. Endo-1,4-β-glucanase is one of the three cellulolytic enzymes in a triplet catalytic system that required for extracellular cellulose hydrolysis. In this study, the sampling was performed from four hot springs in north and northwest of Iran and the screening and identification of acid-stable and thermostable of endo-1,4-β-glucanase producing bacteria were investigated. Endo-1,4-β-glucanase of the isolated strains were determined by qualitative Congo-Red staining as well as quantitative Carboxymethylcellulose/Dinitrosalicylic acid methods as indicators of cellulase activity. Three isolates out of twelve initially selected bacteria, showed noticeable endo-1,4-β-glucanase activity, including Paenibacillus sp. ASh4 , Bacillus sp. AGh1 and Bacillus sp. AG2 with 90%, 77% and 45% residual activity at pH=4 and 60oC after three hours. Molecular identification of the bacteria was carried out using 16S rDNA partial sequencing, in which two isolates belonged to Bacillus sp. and one to Paenibacillus sp.. The results shown that the isolated acido-thermotolerant Bacillus spp. and Paenibacillus sp. had the capability to produce proper acid-stable and thermostable endoglucanase. These isolate also may be even considering as candidates for satisfactory production for other thermostable metabolites with further biotechnological applications.
- انتشار مقاله: 10-10-1396
- نویسندگان: Hasan Diba,Jafar Hemmat,Mohsen Vaez,Mohammad Ali Amoozegar
- مشاهده