ABSTRACT
Penicillin G acylase was immobilized onto iron oxide nanoparticles coated with polyethyleneimine and then cross linked with glutaraldehyde solution. The FTIR spectrum of immobilized enzyme showed peak at 1648cm-1 which can be attributed to the C=N bonds of Schiff’s base linkage formed between glutaraldehyde and amino group of penicillin G acylase. By considering the FTIR spectrum of nano particle coated with polyethyleneimine, adsorption of penicillin G acylase has taken place and then glutaraldehyde cross linked enzyme onto activated support. Catalytic properties of nano penicillin G acylase were improved upon immobilization as compared to its free counterpart. The optimal pH and temperature were determined to be 7.0, 10.0, 50 and 75ºC for free and immobilized penicillin G acylase, respectively. Thermal stabilities of both nano and free penicillin G acylase were studied .The Km value of immobilized nanozyme was calculated from Lineweaver Burck plot to be 0.23 μM while that of free penicillin G acylase was 0.28μM. In this way nano penicillin G acylase with improved catalytic properties was developed as compared to its soluble counterpart.
ABSTRACT
Due to wide range of application of L-Lysine, as an essential amino acid, in different industries, the demand for this amino acid has been increased and this has been led to the development of research on industrial production of L-Lysine. In this study, we tried to increase the yield of production of L-Lysine by genetic optimization. After inspection of different effective enzymes in Lysine biosynthesis pathway, Diaminopimelate dehydrogenase [EC 1.4.1.16] enzyme was selected. This enzyme would be coded by ddh gene. After chromosomal DNA extraction of C.glutamicum ATCC 21799 and designing of primers, the gene fragment was separated from genome by PCR withpfu DNA polymerase enzyme and was cloned in TAcloning vector for facilitation of cloning and determination of nucleotide sequence. Then, enzymatic digestion of TAcloning vector and pET28a vector were performed by EcoRI and Sail restriction enzymes which their products were ddh gene and linear vector. Ligation reaction was accomplished and for checking the accuracy of ligation, it was transformed into E.coliDH5a and finally in expression host, E.coli BL21 [DE3]. The 1150bp band in PCR products and enzymatic digestion of extracted vectors with EcoRI and Sail, sequencing and SDS-PAGE confirmed the accuracy of cloning. Recombinant bacterial colonies were investigated and confirmed by two methods [PCR and enzymatic digestion]. This study showed significant increased expression rate of DAP dehydrogenase enzyme in this expression vector for the first time
ABSTRACT
Background and Purpose: It is thought that transplantation of islet cells would cure diabetic patients in world. Islet cells transplantation in people suffering from diabetes is of technical interest. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells
Materials and methods: In this process, after collagens digestion of pancreases, islets were isolated and dissociated, then with enzymatic procedure by DNase and trypsin, the islet cells changed in to single cells and these cells were assayed by flow cytometery
Results: Flow cytometery of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion
Conclusion: Purified endocrine islet cell grafts were prepared by pure beta-cells, with or without endocrine non-beta cells. The purified aggregates were devoid of non-endocrine cells and damaged cells