Zinc finger (ZF) nucleases are artificial restriction enzymes. Type IIS endonucleases solve this problem by causing the sticky ends to be part of the gene itself, not the recognition sequence (see figure). Type II restriction enzymes result in a scar when two fragments are ligated together. More information on nicking endonucleases can be found in NEB's article.Īdvancements in restriction enzymes Type IIS endonucleases They also provide the NEBcutter tool, which tests a sequence for endonucleases that only cut once. New England BioLabs supplies 10 different nicking enzymes and has information on 552 nicking enzymes in their REBASE database. In contrast to restriction endonucleases, which cleave both strands of a DNA duplex, nicking endonucleases cleave only one strand, introducing a gap into the hydrolyzed strand. This can be desirable, such as when there is more than one recognition sequence. Partial digestion occurs when the concentration of restriction enzymes is too low. In this way, restriction enzymes can be thought of as a basic immune system. The host's DNA is methylated by methyltransferases, so that foreign DNA gets digested. The 5' phosphates remain attached, potentially allowing self-ligation, so a phosphatase step may be necessary following digestion.Ĭleavage can be prevented by methylating the recognition sequence, on A or C nucleotides. Isoschizomers recognize the same restriction site, but they can cleave in a different pattern-both blunt and sticky, with varying amounts of overhang. number specifies that this enzyme is a type II site-specific endodeoxyribonuclease producing 5'-phosphomonoesters.įor a step-by-step guide to cloning using restriction enzymes, see the Barrick Lab's wiki.Īfter the digest reaction, the recognition site will be cleaved, with either blunt (no single-stranded DNA) or sticky (single-stranded overhang) ends. In addition, type II endonucleases do not require energy from ATP hydrolysis.Īn example type II restriction enzyme is EcoRI (E.C.3.1.21.4). Type II are much more useful in the lab, because they cut DNA close to or within the recognition sequence (4-8 base pairs that exhibit inverted-repeat symmetry-see figure). Image from xray crystal structure of 1ERI. Positive residues hold the backbone in place so that the recognition site can be recognized. Pustaka genom dengan sisipan fragmen ~40 Kb yang membawa gen MTGase telah berhasil dikonstruksi dengan menggunakan fosmid pCC1FOSTM di dalam E.coli EPI300-TIR.EcoRI's restriction site in molecular detail. Berdasarkan sekuen gen 16S rRNA, isolat TTA 02 SDS 14 memiliki kekerabatan terdekat dengan Streptomyces thioleteus dengan homologi 99%. Sekuen parsial gen MTGase dari isolat tersebut memiliki homologi 93 % dengan MTGase dari Streptomyces cinnamoneus. Analisis molekuler dengan PCR membuktikan bahwa isolat tersebut mengandung gen MTGase. Hasil penelitian menunjukkan bahwa penapisan terhadap bakteri Streptomyces spp penghasil MTGase yang diisolasi dari berbagai jenis tanah di Indonesia, mendapatkan satu isolat yang potensial yaitu TTA 02 SDS 14. Untuk itu, penelitian ini dilakukan melalui beberapa tahapan yaitu, penapisan bakteri Streptomyces spp penghasil MTGase, identifikasi isolat penghasil MTGase dan pembuatan pustaka genom dengan mengkloning fragmen DNA 40 kb yang mengandung gen penyandi MTGase. Penelitian ini bertujuan untuk mendapatkan bakteri yang menghasilkan MTGase dan mengklon fragmen DNA penyandinya. Pendekatan teknologi rekayasa genetika sangat diperlukan untuk mendapatkan hasil yang lebih baik sehingga dapat dikembangkan dalam skala industri. Selain itu proses pemurnian cukup sulit karena memerlukan beberapa tahap pemurnian. Produksi enzim Microbial Transglutaminase (MTGase) dari strain liar mempunyai beberapa kelemahan yaitu selain pertumbuhan selnya lambat, produk yang dihasilkan sedikit dan protein yang dihasilkan juga bercampur dengan protein lainnya. 2.3.2.13) merupakan enzim yang mengkatalisis reaksi perpindahan gugus asil antara residu glutamin (Gln) yang berfungsi sebagai donor asil dan residu lisin (Lys) sebagai aseptor yang membentuk ikatan silang (crosslinking) ε-(γ-glutamyl) lisin isopeptida yang menghasilkan ikatan kovalen inter atau intramolekuler yang berikatan silang dengan protein makanan.
0 Comments
Leave a Reply. |