Salah satu contoh bakteri yang tahan terhadap kondisi suhu yang tinggi yaitu Thermus aquaticus merupakan jenis bakteri yang dapat mentolerir suhu tinggi, salah satu dari beberapa bakteri yang thermophilik. Kelompok Ini adalah sumber tahan panas enzim Taq DNA Polimerase, salah satu yang paling penting enzim dalam biologi molekular karena penggunaannya dalam polymerase chain reaction (PCR) teknik amplifikasi DNA

Domain: Bakteri

Kingdom: Bakteri

Phylum: Deinococcus-Thermus

Class: Deinococci

Ordo: Thermales

Genus: Thermus

Species: T. aquaticus

Binomial Thermus aquaticus

Brock & Freeze, 1969

Bakteri ini tumbuh subur pada 70 ° C (160 ° F), tetapi dapat bertahan hidup pada suhu 50 ° C hingga 80 ° C (120 ° F hingga 175 ° F). Ini bakteri adalah chemotroph - itu melakukan chemosynthesis dalam rangka untuk mendapatkan makanan. Namun, sejak kisaran temperatur agak tumpang tindih dengan bahwa dari fotosintesis cyanobacteria yang berbagi lingkungan yang ideal, kadang-kadang ditemukan hidup dalam pertalian dengan negara tetangga, memperoleh energi untuk pertumbuhan dari fotosintesis.

• Enzim dari T. aquaticus aquaticus

T. aquaticus telah menjadi terkenal sebagai sumber enzim tahan panas, terutama "Taq" Polimerase DNA, seperti yang dijelaskan di bawah ini.

• Aldolase

Studi thermophilic ekstrem ini bakteri yang dapat tumbuh di kultur sel pada awalnya terpusat pada upaya untuk memahami bagaimana protein enzim (yang biasanya tidak aktif pada suhu tinggi) dapat berfungsi pada suhu tinggi di thermophiles. Bekukan tahun 1970 dan Brock menerbitkan sebuah artikel yang menggambarkan sebuah tahan panas aldolase enzim dari Thermus aquaticus.

• RNA polimerase

Pertama polimerase enzim diisolasi dari Thermus aquaticus pada tahun 1974, adalah DNA-dependent RNA polimerase, [6] yang digunakan dalam proses Transcription.

• Enzim restriksi Taq I

Sebagian besar mungkin ahli biologi molekular menyadari Thermus aquaticus pada akhir tahun 1970 atau awal 1980-an karena isolasi yang berguna endonuklease restriksi dari organisme ini. [7] Penggunaan istilah "Taq" untuk merujuk ke Thermus aquaticus muncul pada saat ini dari konvensi enzim restriksi memberi nama-nama pendek seperti Sal dan Hin, nama-nama yang berasal dari genus dan spesies dari organisme sumber.

Model molekuler Taq polimerase (PDB)

• DNA polimerase ( "Taq pol")

DNA polimerase pertama kali diisolasi dari Thermus aquaticus pada tahun 1976. Keuntungan pertama yang ditemukan untuk tahan panas (suhu optimal 80 ° C) DNA polimerase adalah bahwa itu bisa terisolasi dalam bentuk murni (bebas dari kontaminan enzim lainnya) daripada bisa DNA polimerase dari sumber lain. Kemudian, Kary Mullis dan peneliti lainnya di Cetus Corporation menemukan bahwa enzim ini dapat digunakan dalam polymerase chain reaction (PCR) proses untuk memperkuat segmen pendek DNA, menghilangkan keharusan untuk menambahkan enzim setelah setiap siklus denaturasi termal dari DNA. Enzim juga klon, sequencing,

dimodifikasi (untuk menghasilkan lebih pendek 'Stoffel Fragmen'), dan diproduksi dalam jumlah besar untuk penjualan komersial. Pada tahun 1989 Ilmu majalah bernama Taq polimerase sebagai yang pertama "Molekul of the Year". Pada tahun 1993, Dr Mullis dianugerahi Hadiah Nobel untuk karyanya dengan PCR.

• Enzim lainnya

Suhu optimum yang tinggi untuk Thermus aquaticus memungkinkan peneliti untuk mempelajari reaksi dalam kondisi yang kehilangan aktivitas enzim lainnyaEnzim lainnya diisolasi dari organisme ini meliputi DNA ligase, Alkaline Phosphatase, NADH oksidase, dehidrogenase Isositrat, Amylomaltase, dan selalu populer Fruktosa 1,6-bisphosphate-Dependent L-laktat dehidrogenase.

• Kontroversi

Penggunaan komersial enzim dari T. aquaticus belum aquaticus tanpa kontroversi. Setelah Dr Brock's studi, sampel organisme yang disimpan di Koleksi Budaya Tipe Amerika, repositori umum. Ilmuwan lain, termasuk yang di Cetus, yang diperoleh dari sana. Sebagai potensi komersial Taq Polimerase menjadi nyata pada 1990-an, yang National Park Service berlabel penggunaannya sebagai "Great Taq Rip-off." Para peneliti yang bekerja di Taman Nasional sekarang diharuskan menandatangani "berbagi keuntungan" perjanjian yang akan mengirim sebagian dari keuntungan kemudian kembali ke Park Service.

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ALPHA AMILASE

Alfa-amilase (bahasa Inggris: alpha-amylase, 1,4-alpha-D-glucan glucanohydrolase, pancreatic alpha-amylase, α-amilase, PA) adalah salah satu enzim yang berperan dalam proses degradasi pati, sejenis makromolekul karbohidrat.Struktur molekuler dari enzim ini adalah α-1,4-glukanohidrolase.Bersama dengan enzim pendegradasi pati lain, pululanase, α-amilase termasuk ke dalam golongan enzim kelas 13 glikosil hidrolase (E.C.3.2.1.1) Alpha-amilase ini memiliki beberapa sisi aktif yang dapat mengikat 4 hingga 10 molekul substrat sekaligus.

Mekanisme kerja :

Alpha-amilase pada umumnya aktif bekerja pada kisaran suhu 25 0C hingga 95 0C.[1]Penambahan ion kalsium dan klorida dapat meningkatkan aktivitas kerja dan menjaga

kestabilan enzim ini.[4] Alfa-amilase akan memotong ikatan glikosidik α-1,4 pada molekul pati

(karbohidrat) sehingga terbentuk molekul-molekul karbohidrat yang lebih pendek.[5] Hasil dari

pemotongan enzim ini antara lainmaltosa, maltotriosa, dan glukosa.[1]

. In the Polymerase Chain Reaction (PCR) scientists use DNA polymerase taken from the bacterium Thermus aquaticus to amplify segments of DNA sequences for DNA fingerprinting and other applications.

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Description and significance

Thermus aquaticus was isolated in 1969 by Brocks and Freeze of University of Indiana. It is a Gram-negative bacteria both motile (presence of a flagellum) or immotile. Comparisons between structures of T. aquaticus and E. coli have shown many similarities linking them to a common ancestor. YT-1 gene extracted from T. aquaticus is structurally similar to RecA from E. coli homologue and comaparisons between Klentaq1, a large fragment of Taq DNA polymerase and the Klenow fragment of E Coli DNA polymerase reveal identical C-termini and very similar N-termini. Discovery of this species of thermophiles primarily came as a surprise to researchers and very soon proved to be an extremely important tool in many fields of sciences including biology, microbiology, genetics, diagnostics, clinical laboratories, forensic and environmental sciences, hereditary studies and paternity testing. The importance of this discovery comes from the high thermostability of the T. aquaticus proteins. The taq polymerase plays an extremely important role in the polymerase chain reaction (PCR). PCR is a process by which one or a few stretches of DNA is amplified using thermal cycling introduced by Kary Mullis in 1984 winning him the Noble Prize in 1993. The enzymes found in the T. aquaticus are able to withstand the heat in the denaturing of the newly formed DNA so strands can separate and act as templates for the next cycle of PCR. Taq polymerase, with an optimum activity at 72-80 degrees Celsius and a half life of 9 minutes at 97.5 degrees Celsius, can replicate 9000 base pairs in less than 10 seconds, and presently still plays an extremely important

role in gaining insight into realm of biotechnology. Thermus Aquaticus has not been associated with any known pathology.

Genome structure

T. aquaticus has a double stranded circular DNA chromosome with a length of 2,338,193 nt., with a replicon type WGS, (Master Wgs), no pseudogenes, 53 structural RNA's, 1982 protein-coding sequences, and a pTT27 plasmid.

Cell structure and metabolism

"T. aquaticus" contains pillus like structures used in conjugation. Twelve genes in 3 loci were found to encode preplin-like proteins essential for natural transformation."T. aquaticus" not only can function at high temperatures but they thrive at elevated temperatures. Optimum growth is seen between 60 and 75°C (could go as low as 35°C to as high as 85°C). The optimal pH ranges from 7.5 to 8.0 generally, but some strains grow between pH 5.1 and 9.5. This organism is a chemotroph using carbohydrates, amino acids, caboxylic acids and peptides for growth. Monosaccharides such as glucose are generally used for carbons sources but sucrose, maltose could be used. (Icelandic stains). Some of the proteins isolated from this organism are: elastin, fibrin and casein. Not all strains can hydrolize all substrates. Membranes of these proteins are remarkably temperature stable and the heat stability of the enzymes and protein-synthesis systems allow them to function efficiently at high temperatures. Many factors contribute to the stability of the proteins:

1) Highly organized hydrophobic interiors 2) More hydrogen bonds and presence of other non-covalent bonds strengthen the protein structures. 3) Larger quantities of amino acids like proline make peptide chains less flexible. 4) Protein folding is aided and stabilized by special chaperone proteins. 5) Some evidence indicates that DNA is stabilized by specialized histone-like proteins. 6) Their membrane lipids tend to be more saturated and more branched and possess higher molecular weight resulting in a higher melting point and in turn more thermostability.

Ecology

T. aquaticus was first isolated in the Great Fountain region of Yellowstone National Park from neutral and alkaline springs in 1969 by Brocks and Freeze. This discovery disproved the previous beliefs that

bacteria could not function properly at higher temperatures. After this discovery, some strains of T. aquaticus were discovered in hot springs in Iceland and hydrothermal vents in other parts of the world. T. aquaticus is sometimes found living in conjuction with other organisms such as cyanobacteria and obtain some of the energy for their growth and metabolism from the neighboring bacterias' photosynthesis.

Application to Biotechnology

Enzymes derived from T. aquaticus have had an incredibly important role in facilitating many aspects of biotechnology correlated with DNA amplification. They enable researchers to study proteins and enzymes under conditions not possible before. This is all due to the thermostability of the proteins and their ability to function at even higher rates at high temperatures. Some of the isolated enzymes and their roles in facilitating applications in biotechnology are as follows:

1) Adolase- 2) RNA polymerase- first polymerase isolated from Taq in1974. 3) Restriction Endonucleases 4) DNA polymerase- isolated in 1976, could be isolated in purer form and later discovered to be used in PCR, for amplifying short segments of DNA (before the discovery of the Taq DNA, enzymes needed to be added after each cycle of denaturing of DNA, but with the use of the Taq DNA polymerase it was not necessary anymore.) One single copy of genomic sequence can be amplified by a factor of more than 10 million(One ng of DNA template up to 35 kb could be amplified from a target DNA molecule present only once in a sample of 105 cells) with high base pair fidelity. This enzyme was soon cloned, sequenced, and produced in mass quantities for commercial sale. 5) Other enzymes with high optimal temperatures allowing researchers to study them in extreme conditions are:DNA ligase, alkaline phosphatase, NADH oxidase, isocitrase, dehydrogenase, amylomaltase and fructose1,6-bisphosphate-dependent L-lactate dehydrogenase.

Besides the revolutionary changes in PCR, ligase chain reaction (LCR), which uses T. aquaticus ligase, can amplify genetic sequences of stretches of DNA that posses a desired sequence million or more times within hours. It can amplify and screen in a single step and screen for mutations simultaneously. LCR is useful in testing for hereditary diseases, revealing hidden infections and distinguishing between drug resistant and drug sensitive strains of viruses and bacteria.

Current Research

"THE GENOMOCS OF DISULFIDE BONDING AND PROTEIN STABILIZATION IN THERMOPHILES"

Recent studies have emphasized the role of disulfide bonds in stabilizing the structure of intracellular proteins of Thermus Aquaticus among some other thermophiles. Previously the popular belief was disulfide bonds are only present in extracellular proteins where they stabilize folded proteins against harsh conditions and are rarely found in the cytosol. The specific protein which seems to be responsible for the formation of intracellular disulfide bonds seems to be protein disulfide oxidoreductase (PDO), which functions as a cytoplasmic protein disulfide-isomerase (PDI) . It has been suggested that eukaryotic PDI, found in the endoplasmic reticulum where it catalyzes isomerization of protein disulfide bonds, has evolved from a protein similar to thermophilic PDO. More research needs to be done on this subject.

Besides thermophiles, elevated intracellular disulfide bonding has been seen in other extremophiles including; halophiles, alkylophiles, acidophiles, and radiation-tolerant organisms. This discovery supports the role of intracellular disulfide bonds in stabilizing proteins in all types of extreme conditions. This study sheds some light on different methods used by organisms to stabilize their proteins to adapt to "exotic" environments. [1](1)

Controversy: After isolation of Thermus Aquaticus, samples of it were deposited in the American Type Cultures Collection (ATCC), a public repository. Other scientists had access to them and were able to do more research. By the 1980's, it became obvious that the potential for commercializing the enzymes from this species would prove to be very high and profitable.

"Presence of Bacterial Phage-Like DNA Sequences in Commercial Taq DNA Polymerase Reagents, Tamara Newsome,1 Bing-Jie Li,1 Nianxiang Zou,1 and Shyh-Ching Lo2(2004)"

In a routine study of amplifying the highly conserved gene sequence of the 16S rRNA, several DNA bands were produced that were different from the expected target sizes. Characteristics of these PCR products and further PCR study revealed they were amplified from commercial Taq DNA Polymerase reagents, apparently contaminated with trace amounts of bacteriophage like dnase. A study USING 16s rrna gene primer sA, a number of unexpected products with sizes ranging from 100bp more than 20 kb, in addition to expected 1.5KB. Site products or secondary products are not uncommon, however this time 3 PCR products, 3a, 3b, 7c, were further studied. They were gel purified, cloned, sequenced and compared to sequences in genbank database. A small portion of DNA sequence on 7C, showed 81% homology to tail fiber gene of pesudemona phage, GH-1 and 88% homology to tail fibers proteins of enterobacterial phage T7. 3A and 3B showed partial homology to tail tubular protein B(72%) and putative DNA ligase (55%) of pseudomonas phage GA-1. To confirm findings, four different primers were designed from phage like DNA sequences of these 3 bands and were studied by PCR to detect phage like DNA. The PCR band found were of expected sizes, so all PCR products were confirmed to be phage like DNA sequences by nucleotide sequencing. This

experiment was repeated with Taq polymerases from two different commercial sources. PCR was performed against deionized water sample with or without UV treatment(which has been shown to reduce false positive signals). And the clone 3A, 3B and 7C DNA sequences were added into each group of reaction tubes to serve as positive control, no exogenous DNA template were added to any of the tubes. Hundred mM tris-HCL buffer with a pH 8.3200 micro molar deoxynucleoside triphosphates, 2.5mM MgCl2 25pmol of each primer and 1.25 Uof Taq polymerase. Phage like DNA found in two lots from company A and non from company B. Since company also provided MgCl2 solutions and buffers, they were also tested by swapping them with other samples of buffer and MgCl2. Still same results were observed. Presence of DNA contamination in Taq Polymerases were recorded before but most were exogenous bacterial DNA. This study is the first to show bacteriophage like DNA present. Scientist and Researchers were made aware of these findings. Using tainted amplification reagents can lead to misleading and confusing results. [2]

."PATENT RULING COULD CAUSE PCR ENZYME PRICES, Dalton, R (2001)"

Hoffman La Roche, a Swiss-based pharmaceutical company, owns the patent rights for the Taq DNA polymerase enzyme and the National Parks system was not receiving any of the profits, even though the organism was discovered at a national park. The National Park Service called this "the great Taq ripoff". Since then, researchers in the national parks are required to sign an agreement of benefit sharing so a portion of the profits would be returned to the parks. Meanwhile, a fight for patent rights of the Taq polymerase is still going on. The European Patent office revoked Hoffman La Roche's patent claiming Taq Polymerase is naturally occurring and finding this enzyme was not a "novel invention".

"EUROPEAN PATENT FOR PCR ENZYME CLOUDED BY RUSSIAN CLAIM, Dickson, D (1993)"

Swiss pharmaceutical company acquired the patent rights for the Taq polymerase enzyme from the US Biotechnology company ; Cetus Corporation.While La Roche is fighting the European Patent Office to keep the patent rights, a Russian scientist named Stanislav Gorodetsky is claiming he and his research group were first to discover the organism "T. aquaticus" and isolate the enzyme and they should be involved in the profit sharing. Hoffman La Roche is insisting Russian enzyme is not identical.