SCREENING OF THERMOPHYLIC MICROORGANISM FROM IJEN CRATER BANYUWANGI ASPHYTASEENZYMEPRODUCER . Screening Mikroorganisme Termofilik Penghasil Enzim Fitase yang Tumbuh Di Kawah Ijen Banyuwangi

ABSTRACT Phytase is enzyme which hydrolysis phytic acid to anorganic phosphate and myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphate. The use of phytase in feed industry can overcome.environment and nutrition problems which were arisen from unmetabolismphytic acid or its salt by poultry, swine and fish. The feed industry needs a thermostable enzyme due to the need of high temperature in pelleting process, i.e. 81 ac. By using thermostabile phytase, the pelleting process will not affect the enzyme activity. Thermostabile phytase can be isolated from microorganism live in hot spring water or volcano crater. In this study, the screening of thermophylic microorganism having thermostabilephytase activity in Ijen Crater, Banyuwangi,has been done. From this process, it was obtained 33 isolates that produce phytase enzyme. Isolate was code by AP-17yields highest phytase activity, that is 0.0296 U/mL, so this isolate was choosen for further study. The activity of crude phytase enzyme was measured based on the amount of anorganic phosphate that was produced in enzymatic reaction using UV-VIS spectrophotometer at 392 nm. Based on morphology test to identify the gram type of microorganism, isolate AP-17 has a bacill cell type and identified as positive gram bacteria. This isolate was assumedas Bacillus type. Keywords: Phytase, thermophilicmicroorganism,phytase activit

Cloning, Sequencing and Characterization of The Xylan Degrading Enzymes from Geobacillus thermoleovorans IT-08

Geobacillus thermoleovorans IT-08 is a Gram positive, thermophilic bacterium that can utilize xylan as a sole source of carbon. This strain was isolated from Gunung Pancar hot spring, Bogor, West Java, Indonesia. A plasmid genomic library in Escherichia coli DH5α was constructed and screened for xylanase activity. One positive clone, namely DH5α (pTP510) has been isolated, sequenced and showed putative exo-xylanase (exo-xyl), β-xylosidase (xyl), and α-L-arabinofuranosidase (abfa) genes (Genebank Accession No.DQ387047, DQ345777 and DQ387046 respectively). Each gene encoded 604, 511 and 502 amino acids, respectively. The BLAST search for protein database revealed that Abfa was high similar with GH51 family Abfa of Geobacillus stearothermophilus T6, but Xyl and Exo-Xyl were slight similar with GH43 family (25-34%) respectively. The deduced protein had a molecular weight of about 70 kDa (Exo-Xyl), and 60 kDa (Xyl and Abfa). These showed good accordance with the calculated molecular weight of each protein (68.64 kDa for Exo-xyl, 57.99 kDa for Xyl and 57.03 kDa for Abfa) from deduced amino acid sequence

beta-Xylosidases:Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, beta-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the beta-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, beta-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of beta-xylosidases, and discuss their inhibition by monosaccharides

PEMBENTUKAN SEL R.hQ (SACCHAROMYCES CEREVISIAE) GALUR BARU YANG MAMPU MENCERNA PATI SECARA LANGSUNG MENJADI ETANOL MELALUI KLONING GEN AMILASE

Penyisipan gen pengkode amilase ke dalam ragi Saccharomyces cerevisiae ~. mampu meningkatkan fimgsi dan keIja ragi S. cerevisiae daJam mencerna substrat yang lebih murah, yaitu pati secara Iangsung menjadi etanol. Untuk mencapal tujuan tersebut di atas, maka penelitian ini dilaksanakan selama 3 tahun. Pada tahun pertarna (HB VII 199611997) dan tahun kedua (HB V/2 1997/I998) telah dihasilkan 61? transforman dalam Escherichia coli DHSC/.. Selain itu, pada tahun kedua telah pula dihasilkan amplikon lokus gen amlJase (ALP1) yang berukuran sekitar 1400 ph. Amplikon tersebut telah dikarakterisasi dan diinsersikan ke dalam vektor T· pMOSBlue. Pada tahun ketiga penelitian ini, dilakukan analisis adanya insersi gen amilase dalam transforman E.coli DHSa. dengan teknik Direct Screening peR. Kondisi amplifikasi peR yang digunakan sarna dengan kondisi peR pads tahun kedua, yaitu denaturasi pada suhu 11 ADLN - PERPUSTAKAAN UNIVERSITAS AIRLANGGA LAPORAN PENELITIAN PEMBENTUKAN SEL RAGI... PUSPANINGSIH, NI NYOMAN T. 94ne selama I menit, annealing pada suhu 50ne selama I menit dan ex/en/Ion pada suhu 72Cse1ama2menit. Jurnlahsiklus adaIah 30 dengan I kali pemantapan ex/en/ion selama 4 menit. Sebagai DNA templat adalah DNA total daIam transforman E.coli DH5a. Sebagai kontrol positif adalah pSFaI dan amplikon (hasil tahun kedua). Selanjutnya, koloni transforman positif dianalisis Iebih Ianjut, meliputi isolasi dan karakterisasi klon serta uji ekspresi ami1ase dengan metode Nelson Somogyi. ....... Hasil Direct Screening peR menunjukkan adanya 3 koloni transforman yang diduga mengandung insert gen amilase. Namun hasil isolasi DNA plasmid dalam skala kecil menunjukkan bahwa ketiga koloni tersebut mengandung DNA rekombinan yang-sl:lfIill.-Selanjumya basil karakterisasi DNA rekombinan tersebut dengan enzim restriksi StuI, HindI dan EcoRl menunjukkan urutan nukleotida daerah insert. Pernotongan dengan enzim StuI menghasi1ak 3 pita yang berukuran ± 5700, ± 3000 dan ± 700 pb.; enzim HindI . menghasi1kan 4 pita yang berukuran ± 3200, ± 2300, ± 2100 dan ± 1800 pb dan EcoRl rnenghasilkan 2 pita yang berukuran ± 700 pb (diduga pada daerah insert) dan ± 8700 pb (diduga pada daerah vektor asaJ YCp50). Pemotongan dengan enzim restriksi . ClaI hanya memberikan 1 pita yang berukuran ± 9400 pb. Ukuran ini lebib besar dan ukuran vektor asa1nya YCp50, sehingga diduga ukuran insertnya adaJah ± 1500 pb. Transforman ini menunjukkan aktivitas amilase sebesar 88,0265 Unit dengan metode Nelson-Somogyi pada A= 748 DID. Basil ini Jebih besar dibandingkan kontrolnya

Peningkatan aktivitas enzim hemiselulase dengan rekayasa protein untuk pengolahan biomassa berbasis lignoselulosa

The purpose of this research is to investigate the substrate stereospecificity of IlL- arabinofuranosidase GH51 Geobacillus thermoleovorans IT-08 (AbfA) toward Mycobacteria cell wall. In general, there are 3 stages in this research are (1) the production of AbfA recombinant from E. coli BL21 (DE3)/pET-abfA, (2) specific activity assay of AbfA recombinant, (3) substrate stereospecificity analysis of AbfA in silico. In laboratory methods, AbfA has hydrolase activity toward Darabinofuranoside (H37Rv and BFCC). However its activity toward Larabinofuranoside (arabinogalactan, pectin, oat spelt xylan and arabinan) higher than its hydrolase activity toward D-arabinofuranoside (H37Rv and BFCC). On silico analysis, hydrolase activity AbfA toward D-arabinofuranoside may occur due to fingerprint interactions between the ligand and catalytic residue Glu294. Based on in silico analysis, the catalytic mechanism of AbfA toward D-arabinofuranoside was suggested following model catalytic mechanism of a-L-arabinofuranosidase GHSI toward L-arabinofuranoside substrate. Effect of sterie hindrance by Trp99 and Trp298 at the sub-site -1 rationalize the substrate specificity toward D-arabinofuranoside lower than L-arabinofuranoside. This thesis research was the first reported that AbfA has catalytic activity toward D-Arabinofuranoside derived from Mycobacteria cell wall

Purification of β-1,3-Endoglucanase from Cabbage (Brassicaoleracea cv. capitata L.) by Ion Exchange Chromatography

In this research, there was purified of β-1,3- endoglucanase from cabbage, having the aim to calculate purification level of enzyme by ion exchange chromatography methods. That process was started by producing enzyme isolated from Gloria osena hybrid of cabbage,then precipitate it using ammonium sulphate with concentration of saturated 40%. The sediment of enzyme was diluted by phosphate citrate then purified by dialysis in order to separate enzyme from other proteins and ammonium sulphate. The next, hydrophobic interaction chromatography eluted by ammonium suphate concentration of saturated 40% in Tris HCl pH 7 (high concentration to low concentration gradient) with Butyl-topearl 650M in ethanol as matrix was done to purify enzyme based on hydrophobic group interaction of protein and absorbent. After that, there was purifying enzyme by ion exchange chromatography in order to separate enzyme based on it’s ion. Enzyme was eluted by NaCl (0- 0.5) M in Tris HCl (low concentration to high concentration gradient) with DEAE-toyopearl 650 M in ethanol as the matrix. The best result of this ion exchange chromatography on first fraction pH 7 had purification level 3.534,1 of initial extract

Pemetaan Biodiversity Bahan Limbah Agroindustri Untuk Formula Pakan Komplit Menggunakan Enzim Lignoselulolitik Dalam Meningkatkan Ketahanan Pangan

Potency produce waste of agroindustri high enough, but that way problems that happened is lowering of value of nutrition marked with obstetrical height of crude fibre cellulose, hemicellulose, lignine) and lower crude protein. Cellulose and hemicellulose available only some of small able to be exploited is most castaway become waste. Cellulose molecule and of hernicelulosa represent polysaccharide with tying 13- 1-4 glicosidic which is difficult to be digested by bacterium of rumen livestock of ruminantia, so that waste digesting of agroindustry become to lower. Usage of enzymes lignocellulolytic will be able to improve value of nutrition. Enzyme of cellulase consist of three enzyme component there are: endoglucanase ( endo-I,4-J3- D-Gluconase, or 1,4-D- Glucan 4-glucanohydrolase), eksoglucanase ( exo-I,4-J3-D-Glucanase, or 1,4- - Cellobiohydrolase D-glucan) and cellobiase ( - or glucosidase - Glucohidrolase Dglucosedi), while enzyme of xylanase consist of five enzyme component there are endoJ3- 1,4-xylanase, J3-xylosidase, - L-Arabinofuranosidase, - D-Glukuronidase, and acetyl xylan esterase. The aim of this research were exploration lignocellulolytic enzymes (cellulase and xylanase) from bacterium of rumen beef cattle, characterization optimum pH and temperature, determine molecular weight of protein cellulose and xylanase enzymes, and purification level of enzymes by diafiltrate and ion exchange chromatography. In this research, identification with Carboxyl Methil Cellulose (CMC) and Oat Splet Xylon has been done. The result showed that isolate BR2 have activity of endo-l,4-J3-D-Gluconase 0,28 Ulm and endo-B-I,4-xylanase 22,63 U/rnl. The optimum pH and temperature of celulase and xylanse were 6, 7 , 45°C, 50 °C. This process was started by producing cellulase and xylanase enzymes from isolate BR2, then precipitated it using ammonium sulphate with optimum concentration of saturated 40% and 60%, and the molecular weight protein of cellulase and xylanase enzymes were 49 kDa and 80 kDa. Based on experiment that celluloliytic enzymes from BR2 could hydrolyzed several specific substrate (CMC, pNPG dan pNPC), with activities crude extract cellulolytic are 3,51 UII, 1,86 UII dan 2,5 UII. Based on experiment that xylanolitic enzymes from BR2 could hydrolyzed several specific substrate (Oat spelt xi/an, pNP-X, pNP-G, pNP-acetyl acid, v pNP-A ), with activities crude extract xylanolitic were 0,433 U/ml, 0,034 U/m!, 0,018 U/ml, 0,008 U/ml, 0,0 II Ulm!. The best result of this diafiltrate on pNPG and pNPacetyl acid had level 8,01 and 49,90 from crude exctract. Beauchemin, K.A., D. Colombatto, D.P. Morgavi and W.Z. Yang. 2003

PENINGKATAN AKTIVITAS ENZIM HEMISELULASE DENGAN REKAYASA PROTEIN UNTUK PENGOLAHAN BIOMASSA BERBASISLIGNOSELULOSA

LAPORAN PENELITIAN INI MEMBAHAS TENTANG PENINGKATAN AKTIVITAS ENZIM HEMISELULASE DENGAN REKAYASA PROTEIN UNTUK PENGOLAHAN BIOMASSA BERBASISLIGNOSELULOS

Enhanced production of thermophilic xylanase by recombinant Escherichia coli DH5a through optimization of medium and dissolved oxygen level.

Enhancement of thermophilic xylanase production by recombinant Escherichia coli DH5α through suitable medium formulation was initially investigated using shake flask cultures. Thereafter the effect of dissolved oxygen tension (DOT) level on the performance of xylanase fermentation by E. coli DH5α was investigated in 2 L stirred tank bioreactor using the optimal medium. Among the two basal medium tested (complex medium of Luria Bertani & defined mineral medium), defined mineral medium gave the highest growth and xylanase production. The optimal glucose and (NH4)2504 for xylanase production was obtained at 10 g L -1 and 2 g L-1, respectively. Growth of E. coli DH5α and xylanase production was inhibited in oxygen limited fermentation, where dissolved oxygen tension level was controlled at 0% saturation. On the other hand,xylanase production was enhanced at DOT level controlled at 20% saturation, though growth was not significantly improved. Substantially high xylanase production (1784.57 U mL -1) was obtained in fermentation using optimal medium composition and DOT level. These results indicate that efficient process control strategy is important for the mass production of xylanase enzyme by E. coli DH5α