Production of Cellulase Enzyme from Aspergilus niger using Rice Husk and Bagasse as Inducer

Aspergillus niger is fungi can produce cellulase enzyme with agriculture waste as natural inducers. The purpose of this study was to compare the natural inducers potential between rice husk and bagasse to produce cellulase enzyme from Aspergillus niger. Production of cellulase enzyme was done with variety of inducers such as CMC, rice husk, and bagasse. The optimization of enzyme production includes optimum production time, inducer type, and optimum concentration of inducer. Furthermore, the enzyme also was characterized in pH and temperature. Enzyme activity test using the DNS method with CMC as substrate. According of this test result show that highest cellulase enzyme activity has production time for 108 hours with rice husk as inducer. The optimum rice husk concentration was needed of 2.5%. The cellulase enzyme was induced by rice husk has optimum activity at pH 4 and 50°C of 0.709 IU/mL.   Keywords : cellulase enzymes, Aspergillus niger, inducers, rice husk, bagasse

Pencirian Produksi Amilase oleh Saccaromyces Cerevisiae W303A Rekombinan

Characterization of Amylase Production by Saccharomyces cerevisiae W303A Recombinants. Cloning of amylase gene from Endomycopsis fibuligera ITB.R.cc.64 into S. cerevisiae W303a can effectively increase the yeast function to digest starch directly into ethanol. Production of amylase by S. cerevisiae W303a recombinants (I and P) were done by growing in yeast peptone starch (YPS) medium. The result showed that the recombinants could be produced of amylase by gave clear zone after staining by iodium vapor. The optimum condition of production of amylase by S. cerevisiae W303a recombinants were pH 7.0, 40?C temperature incubation, and gave maximum activity after 36 hours incubation. Amylase activity of I was higher than P recombinant for these condition respectively

Constructing and Screening Beta-Glucanase Activity of Metagenomic cDNA Expression Library of Digestive Gland of Achatina fulica

Digestive juice of Achatinafulica shows hydrolase activities which are useful as an antibiofilm. Insidethe digestive system of snail many microorganisms live together as a normal flora.In this study, a metagenomicbased-activity technique was carried out to construct the metagenomic expression library of digestive gland ofAchatinafulica. The metagenomic library was screened for the recombinant harbouring genes encoding beta-glucanase again the specific substrate of laminarin.The metagenomic cloning was carried out by using λTriplEx2 as vector and E.coli XL1-Blue as host. The longer than 0.5 cDNAs ligated to vector and the total ligation solution were 1000 μL.Circularization of each recombinant phage inserted the target sequence were conducted in E.coli BM25.8, to become recombinant phagemids. Recombinant phagemids screening were carried out by using restriction method to analyse the DNA insert.We produced 2.8x108 plaques and 1.1x1010 library amplification titer. Seventeen hallo plaques from 100 μL of the ligation solution positive screening with laminarin substrate.Eight recombinant phagemids were randomly picked and analysed using restriction method showing different fragment. A high quantity cDNA library from digestive gland Achatinafulica was successfully constructed

Shortening of Amino Acids from C-terminal of PZase as Basis of Pyrazinamide Resistance in P14 Isolate of Mycobacterium Tuberculosis Strain

AbstractPyrazinamide (PZA) is one of the mainstays WHO-recommended drugs for therapy of tuberculosis (TB). The emergence of PZA resistance in clinical isolates of M. tuberculosis is often associated with pncA gene mutations encoding PZase. A local clinical isolate of Mycobacterium tuberculosis strain showed phenotipe resistant to PZA at concentration of 10μg/mL. The ORF of pncA gene of the isolate showed deletion of guanine base at position 81, then followed by shortening of 70 amino acids from C-terminal of PZAse which has 186 amino acid residues. The mutant of PZase took frame shift of amino acids after the residue at position 27. The pncA gene mutation at the level of genotype, that produced a physical-chemical alteration of the active site or the metal-binding site of PZase, in this case perturbing or lossing its activity was proposed as trigering the PZA resistance in P14 clinical isolate of M. tuberculosis strain

Immunogenicity assay of KatG protein from Mycobacterium tuberculosis in mice: preliminary screening of TB vaccine

the tuberculosis (tB) disease is still widely found even though BCg vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. objective of the research was to determine the immunogenicity of katg protein of M. tuberculosis clinical iso-late L19 in mice. the katg protein as antigen was prepared by expression of the katg gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the katg was vaccinated to mice to detect its immunogenicity. the expression of katg in e.coli BL21 could result in katg protein with molecular weight 80 kda in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of Igg1, Igg2a, Igg2b, Igg2c, Igg3, and IgM. the highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. the katg protein exhibited a high immunogenici ty in mice, so it is possible to develop as a vaccine candidate for tB. a clinical test should be performed in a future to ensure its safety as a therapeutic protein

Structure-Activity Relationship of Mutant KatG from INH resistant Mycobacterium tuberculosis

Mutation in katG gene of Mycobacterium tuberculosis encoding catalase-peroxidase that damage its enzyme activities is well associated with isoniazid (INH) resistance. The katG gene from INH resistant strain of M. tuberculosis clinical isolate L19 has been observed in previous study, carrying mutations of G 234 A and C T,and changed the arginine residue at position 209 with cysteine in its KatG protein. However the activities of the mutant protein has been not known yet. Expression of the katG gene L19 that was done in Escherisicia coli BL21(DE3) using pCold II-DNA generated KatG protein with 80 kDa in SDS PAGE electroforegram. The mutant protein of KatG L19 decreased 43% of catalase activity and 11% of peroxidase activity against to KatG wild type (H37RV). The model structure of the mutant KatG protein had deviation structure toward KatG wt as 0,13 for number of Root Mean Square Deviations (RMSD). The mutant KatG (Arg209Cys) losed two hydrogen interactions and a van der Waals bond which present in KatG wild type. In the KatG wt protein, the both hydrogen bonds was formed between the Arg209 residu to Glu201, while the van der Waals bond occured by lingking of Arg209 residu to Glu217. Disruption in the some chemical interactions might trigger the decline in catalase-peroxidase activities of mutant KatG L19 and further it bring out the INH resistance in the clinical isolate L19

Mekanisme Resistensi Isoniazid Pada Mycobacterium tuberculosis: Analisis Mutasi Gen katG dan Kaitannya Dengan Sifat Biokimia Enzim Katalase-Peroksidase Mutan Rekombinan

Tuberkulosis (TB) merupakan penyakit infeksi menular yang disebabkan oleh Mycobacterium tuberculosis dan menyebabkan kematian banyak penduduk dunia. Resistensi M. tuberculosis terhadap obat dapat mempersulit pengobatan TB. Multidrug resistant (MDR) TB merupakan galur M. tuberculosis yang resiten terhadap minimal dua obat TB Iini pertama, yaitu isoniazid dan rifampisin. MuncuInya sifat MDR dapat dihasilkan dari akumulasi mutasi gen-gen yang menjadi target obat anti TB. Beberapa isolat klinis M. tuberculosis menunjukkan resisten isoniazid, tetapi penyebab rensistensi tersebut masih belum diketahui. Penelitian ini bertujuan utama untuk mengungkap penyebab dan mekanisme resistensi isoniazid pada isolat klinis yang dimaksud melalui penentuan mutasi gen katG pada tingkat genotipe dan karakterisasi enzim katalaseperoksidase mutan pada tingkat protein; serta mempelajari keterkaitan antara mutasi gen katG dan aktivitas katalase-peroksidase mutan dengan tingkat resistensi isoniazid. Pada tahun pertama ini. penelitian telah diarahkan untuk mengidentifikasi keragaman mutasi gen katG pada koleksi isolat-isolat klinis dan menganalisis kaitan antara mutasi gen tersebut dengan tingkat resistensi secara in silico. Tahapan keIja pada penelitian tahun pertama terdiri atas amplifikasi gen katG M. tuberculosis isolat klinis dan wild type, cloning gen pada vector pGemT, anaIisis restriksi DNA rekombinan pGemT-katG dan sekuensing gen katG, analisis mutasi, subldoning gen katG pada vector pET20b, dan resekuensing gen katG. Gen katG M. tuberculosis isolat klinis resisten isoniazid (L8, L19, LI0, L21, R2 dan R9) yang berukuran 2,2 kb berbasil diamplifikasi dengan PCR menggunakan pasangan primer FG dan RG. Karakterisasi molekuler gen katG melalui tahapan kloning dan subkloning dapat menunjukkan adanya varian nukleotida pada gen kalG isolat-isolat klinis tersebut. Mutasi nukleotida yang ditemukan pada gen kalG isolatisolat klinis ditengarahi sebagai penyebab munculnya resistensi isoniazid. Hasil translasi in silico terhadap urutan nukleotida gen kalG isolat-isolat klinis yang dipelajari menunjukkan bahwa mutasi asam amino pada bagian domain N protein KatG lebih cenderung meningkatkan efek resistensi isoniazid daripada mutasi yang terjadi pada bagian domain C. Mutasi yang mengubah residu asam amino yang dekat dengan residuresidu kritis protein KatG cenderung lebih meningkatkan efek resistensi daripada mutasi asam amino yang letaknya jauh dari sisi kritis. Mutasi yang mengubah residu amino dikedua domain N dan C protein KatG lebih cenderung meningkatkan efek resistensi isoniazid daripada mutasi pada salah satu domain. Makin banyak jumlah mutasi yang terdapat pada gen kalG, kecenderungan meningkatkan tingkat resistensi isoniazid juga semakin tinggi. Perlu dilakukan penelitian lanjutan yang mengungkap aspek aktivitas enzim katalase-peroksidase M. tuberculosis terkait dengan ekspresi, pemumian dan karakterisasi protein KatG. Hal ini menjadi penting dalam upaya untuk mendapatkan konsep mekanisme resistensi isoniazid secara lebih komprehensi

RESISTANCE LEVEL OF Pseudomonas stutzeri AGAINST MERCURY AND ITS ABILITY IN PRODUCTION OF MERCURY REDUCTASE ENZYME

Mercury reductase is an enzyme that is able to reduce Hg2+ to Hg0 non toxic. This enzyme is usually produced by mercury resistant bacteria. The research wanted to determine the resistance of indigenous Pseudomonas stutzeri isolate toward mercury and to explore the mercury reductase activity which is produced by the bacteria. The results of resistance assay of the Pseudomonas stutzeri toward mercury ion showed that the isolate could survive in media containing HgCl2 up to a concentration of 80 µM. The bacteria could produce mercury reductase optimally at the 24th of fermentation time. The enzyme showed optimum activity at pH 7 and temperature of 45 o

FRAKSINASI EKSTRAK KASAR AMILASE DARI Endomycopsis fibuligra UNTUK MEMISAHKAN SACCHARIFYING DAN LIQUEFYING AMYLASE

Telah dilakukan penelitian tentang fraksinasi ekstrak kasar amilase dari Endomycopsis Fibuligera untuk memisahkan Saccharifying dan Liquefying Amilase. Fraksinasi dilakukan dengan amonium sulfat, yang konsentrasinya dinyatakan dalam prosen jenuh. Prosen jenuh amonium sulfat yang ditambahkan pada ekstrak kasar amilase adalah 40 % , 60%, dan 80 %. Hasil penelitian menunjukkan bahwa aktifitas sakarifikasi dan likuifikasi pada ekstrak kasar amilase dapat terpisahkan oleh fraksinasi tersebut dan dihasilkan empat fraksi amilase yaitu fraksi 0-4%, 40-60%, 60-80% dan supernatan. Aktivitas likuifikasi murni pada fraksi 0-40%, 40-60%, sakarifikasi murni pada fraksi 40-60% dan aktivitas kedua-duanya didapat pada fraksi 60-80% dan supernatan

Kloning Dan Over Ekspresi Gen Protein Disulfida Isomerase Ragi Saccharomyces cerevisiae DI Escherichia coli

Protein disulfida isomerase (PDI, E.C.5.3.4.1) yang dikode oleh gen PDII ragi adalah suatu enzim yang berfungsi mengkatalisis reaksi pembentukan dan penataan ulang (isomerisasi) ikatan disulfida dalam proses folding polipeptida suatu protein ekstraselular atau protein jalur sekresi. Selain itu juga merupakan molekul chaperon yang membantu proses pelipatan protein di dalam retikulum endoplasma, dengan cara menghambat pembentukan ikatan disulfida selamafolding polipeptida sehingga pembentukannya tidak terjadi secara random. PDI mengarahkan pembentukan ikatan disulfida tersebut dapat terjadi diantara pasangan residu sistein yang tepat, sehingga menghasilkan molekul protein dengan struktur tersier yang native. PDI juga merupakan komponen dari dua enzim multimer prolil-4-hidroksilase dan protein kompleks pentransfer trigliserida mikrosom (Freedman et a1.,1994). Fungsi PDI sebagai enzim dan molekul chaperon, mendasari aplikasi PDI dalam bidang kedokteran, industri makanan, farmasi maupun industri kimia lainnya. Penggunaan PDI yang beragam menuntut penyediaannya dalam skala besar. Ragi Saccharomyces cerevisiae menghasilkan PDI kurang dari 0.05 % dari total protein sel (Mizunaga et a1.,1990). Rendahnya kadar PDI ragi ini memerlukan upaya over produksi PDI sehingga ketersediaanya dapat memenuhi kebutuhan di banyak industri. Penelitian ini dirancang untuk melakukan over produksi enzim protein disulfida isomerase (PDI) ragi Saccharomyces cerevisiae di Escherichia coli. Penelitian ini akan diawali dengan mengamplifikasi dan mengklon gen protein disulfida isomerase ragi (PDII)di Escherichia coli DH5oc. Permasalahan penelitian pada tahap awal ini adalah : apakah gen PDI ragi dapat diamplikasi dengan PCR, berapakah ukuran molekul gen PDI1, apakah penyambungan gen PDI1 ke vektor pGemT dapat menghasilkan rekombinan pGemT-PDII, dan apakah rekombinan tersebut dapat diklon di Escherichia coli DH5a. Penelitian tahap awal ini bertujuan untuk mengamplifikasi gen protein disulfida isomerase dengan PCR, menentukan ukuran gen hasil PCR, dan mendapatkan rekombinan pGem-T-PDII dalam klon Is. co/i D1-15a. Kloning gen Protein disulfida isomerase ragi di E. coli DH5a dikerjakan dengan cara mengamplifikasi gen PDI ragi menggunakan PCR. Amplikon hasil PCR selanjutnya diligasi dengan vektor kloning (plasmid pGem-T) untuk mendapatkan DNA rekombinan pGem-T-PDII. DNA rekombinan hasil konstruksi ini, kemudian digunakan untuk menstransformasi E. coli DH5a. Seleksi transforman dilakukan untuk mendapatkan klon E. coli DH5a [pGemT-PDII]. Amplifikasi gen PD11 ragi dengan dua buah primer, yaitu primer forward dan primer reverse menggunakan PCR telah berhasil mendapatkan suatu fragmen DNA berukuran ± 1700 pb. Sementara itu, ligasi amplikon hasil PCR dengan vektor T (pGem T) dapat menghasilkan rekombinan pGemT-PDII di dalam Escherichia coli DH5a. Karakterisasi pemotongan rekombinan pGemT-PDII dari beberapa transforman E.coli dengan enzim restriksi Ndel dan 13amHI menunjukkan bahwa gen PD11 telah berhasil terklon di E. coli DH5a. Kesimpulan dari penelitian ini adalah bahwa fragmen DNA berukuran ± 1700 pb yang ekuivalen dengan ukuran gen PD11 dapat dihasilkan dari amplifikasi gen PDIl ragi menggunakan PCR. Sebuah fragmen DNA (± 4700 pb) hasil pemotongan pGem T rekombinan oleh enzim tunggal Ndel, dan BamHI, serta dua fragmen DNA (± 3000 pb) dan (± 1700 pb) hasil pemotongan oleh dua enzim Ndel dan BamHI, menunjukkan bahwa rekombinan pGemT-PDII telah terkontruksi dan berhasil terklon di E. coli DH5a. Saran penelitian ini adalah perlunya melakukan penentuan urutan nukleotida gen PD11 yang telah diamplifikasi dan juga meng-over ekspresikan gen tersebut baik di E.coli maupun di ragi