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"Keberhasilan produksi apoptin rekombinan dalam bentuk native pada penelitian sebelumnya (Khalid, 2012) membuka jalan untuk mengembangkan produksi protein antikanker ini ke skala yang lebih besar. Di dalam studi ini, dilakukan optimasi kultivasi bakteri rekombinan apoptin dalam stirred tank fermentor dan bakteri yang digunakan adalah bakteri Bacillus subtilis 168 rekombinan apoptin hasil transformasi dengan sistem Gateway menggunakan plasmid pOXGW12His8Arg. Parameter yang dioptimasi adalah konsentrasi induksi xylose, kecepatan agitasi dan laju aerasi. Variasi konsentrasi induksi xylose dilakukan dalam shake flasks dengan volume kultur 100 ml dengan konsentrasi 0-5% b/v sedangkan variasi kecepatan agitasi dan laju aerasi dilakukan dalam stirred tank fermentor dengan volume kultur 3L dengan kecepatan dan laju masing-masing adalah 150-250 rpm dan 0,5-1,5 NL/min. Hasil yang didapat adalah pertumbuhan bakteri optimum dicapai pada konsentrasi xylose 1% b/v, kecepatan agitasi 250 rpm, dan laju aerasi 1,5 NL/min dengan nilai laju pertumbuhan spesifik bakteri untuk masing-masing variasi adalah 0,628 h-1; 0,630 h-1; dan 0,747 h-1.

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The success of recombinan apoptin production in native form in the previous research (Khalid, 2012) open the way to develop this anticancer protein production to the larger scale. In this study, optimization of recombinant apoptin bacteria cultivation is carried out in a stirred tank fermentor using Bacillus subtilis 168 with plamid pOXGW12His8Arg which transformed by Gateway method. The optimized parameters are xylose-inducer concentration, agitation speed, and aeration rate. The xylose-inducer concentration variation is carried out in a shake flasks with 100 ml volume broth, while the agitation speed and aeration rate variation is carried out in a stirred tank fermentor with 3L volume broth. The xylose concentration is varied between 0-5% w/v, while agitation speed and aeration rate are varied between 150-250 rpm and 0,5-1,5 NL/min respectively. The best condition in this cultivation is 1% w/v of xylose, 250 rpm of agitation speed and 1,5 NL/min of aeration rate giving the specific growth rate value for each parameter of 0,628 h-1; 0,630 h-1; dan 0,747 h-1 respectively."

"ABSTRACTXylitol is a five-carbon polyol sugar. It has many healthy benefits and is widely used in food, pharmaceutical, and healthcare. Natural sources with abundant carbon such as lignocellulose can be used for xylitol production. One of the potencial sources with high prevalency in Indonesia is water hyacinth. It is known as weeds and has not been fully utilized by people. The aim of this research was the utilization of water hyacinth which contains hemicellulose as a substrate in the bioconversion of xylose into xylitol by yeast cells Debaryomyces hansenii. Stages of processing include the optimization of water hyacinth hydrolysis using autohydrolysis method and optimization of fermentation conditions. Xylose and xylitol were determined by HPLC with RI detector and LiChrosorb® NH2 (4 mm x 125,00 mm, 5μm) column. Acetonitrile-water (90:10, v/v) was used as a solvent. 20 μL sample volume was injected at flow rate of 1.0 mL/min and room temperature. The results showed that optimum conditions for the acquisition of xylose were obtained through autohydrolysis methods for 75 minutes with 1:15 water hyacinth and water ratio and posthydrolysis for 45 min using 4% sulfuric acid. Xylose concentration in hydrolyzate obtained was 25.55 g/L. The optimum fermentation condition for xylitol production was achieved by four day cultivation, limited aeration condition, and addition of metal ions CaCl2.2H2O 0.01%. The yield of xylitol obtained using those conditions was 77.43 %. "

"ABSTRACTXylitol is five-carbon polyol sugar which widely used as a sweetener in food and pharmaceutical.Xylitol production by chemical procedures using high pressure and temperature also neededextensive purification are less cost-effective in production. Fermentation which has more advantageswith lower cost due tocheaper substrate and the non-necessity of xylose purification. The purposes ofthis research were to find optimum condition for xylitol production with particular variable such assubstrate concentration, aeration, methanol and nitrogen sources addition. Oil palm empty fruitbunch hydrolyzates containing xylose was fermented into xylitol by Debaryomyces hansenii UICC Y-276 at room temperature. Fermentation was carried out at 200 rpm for 72 hours. Then, xylose andxylitol were determined by HPLC with RI detector and LiChrosorb® NH2 (4 mm x 125,00 mm, 5μm)column. Acetonitrile-water was used as a solvent, 20 mL sample volume was injected at flow rate of1,0 mL/min at room temperature. The optimum fermentation conditions was obtained in a state ofsemi-anaerobic condition (1 : 2.5) with 10,0 % (w/v) xylose concentration. Meanwhile with theaddition of various concentration of methanol and nitrogen sources, it was obtained that 1,5 %methanol and 0,5 % ammonium sulfate gave high yield of xylitol production. The best result for yieldxylitol production was 31,83 %."

"ABSTRAKKanker adalah salah satu penyakit mematikan yang pengobatannya terus dikembangkan. Apoptin adalah molekul protein yang berpotensi untuk dijadikan obat kanker karena mempunyai aktivitas menginduksi proses kematian sel secara selektif hanya pada sel kanker saja. Kloning apoptin telah berhasil dilakukan dengan amplifkasi gen menggunakan PCR dengan menambahkan 12-histidin dan 8-arginin pada C-terminal kemudian diligase ke plasmid pOXGW dengan sistem Gateway, lalu diekspresikan ke dalam bakteri Bacillus subtilis 168. Plasmid pOXGW - apoptin - 12His8Arg dapat terekspresi di B. subtilis. Dalam penelitian ini Bacillus subtilis yang membawa plasmid diproduksi pada medium dengan variasi xylose sebagai substrat pemicu dan sebagai pembanding bakteri Escherichia coli Bl21 Star™ ditransformasi dengan plasmid pOGW - apoptin - 12His untuk kemudian dilakukan pemurnian. Hasil penelitian menunjukan apoptin rekombinan dari B. subtilis 168 yaitu 568 μg/ml, sedikit lebih banyak dari jumlah protein rekombinan E. coli Bl21 Star™, 421 μg/ml.

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ABSTRACTCancer is a deadly disease so that the medicinal treatment constantly developed. Apoptin is a protein molecule that has potential to be used as a cancer drug because of its activity to induce cell death selectively to the cancer cells only. Cloning apoptin has been successfully performed by amplify gene using PCR with 12-histidine and 8-arginine to be added at C-terminal then ligated into plasmid pOXGW with Gateway system, and then expressed in Bacillus subtilis 168. Plasmids with pOXGW - apop - 12His8Arg can be expressed in B. subtilis. In this study, Bacillus subtilis carrying plasmid was produced with variations of xylose as substrate trigger on liquid medium and as a comparison, Escherichia coli Bl21 Star™ transformed with a plasmid pOGW - apop - 12His and then performed for purification of apoptin. The results showed that the recombinant apoptin obtain from B. subtilis 168 compared to Escherichia coli Bl21 Star is slightly higher, i.e. 568 μg/ml and 421 μg/ml, respectively."

"Indonesia memiliki ketergantungan terhadap impor kit untuk Polymerase Chain Reaction (PCR). Salah satu komponen krusial dalam PCR adalah DNA polimerase termostabil. Hal ini tidak sebanding dengan Indonesia yang terletak pada Ring of Fire, dimana Indonesia memiliki potensi ekosistem geothermal yang besar sebagai habitat bakteri termofilik penghasil DNA polimerase termostabil. Gen DNA pol I lokal dari Geobacillus thermoleovorans Batu Kuwung, Banten, telah berhasil dikloning pada plasmid pET-15b dan ditransformasikan pada Escherichia coli BL21. Meskipun GBK pol memiliki peran yang sangat penting dalam amplifikasi isothermal yang menjadikannya dapat diaplikasikan dalam PCR, untuk dapat memproduksi GBK pol secara komersil masih dinilai kurang layak secara ekonomi. Penelitian yang telah dilakukan untuk GBK pol masih memiliki keterbatasan dalam optimalisasi dan produksi scale-up. Oleh karena itu, pada penelitian ini dilakukan optimasi kondisi ekspresi seperti: kecepatan agitasi, waktu inkubasi setelah induksi, volume kerja dan peningkatan skala produksi pada 7,5 L bioreaktor untuk meningkatkan efisiensi, produksi dan ekspresi. Pada penelitian ini didapatkan kondisi optimum pada kecepatan agitasi 200 rpm, waktu inkubasi setelah induksi 4 jam dan 20% volume kerja. Selain itu, pada penelitian ini berhasil dilakukan produksi GBK pol pada 7,5 L bioreaktor dan menghasilkan konsentrasi protein tertinggi sebesar 0,042 µg/µL dengan waktu optimum untuk inkubasi setelah induksi selama 3 jam.

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Indonesia relies on imported kits for Polymerase Chain Reaction (PCR). In fact, one of the crucial components in PCR is thermostable DNA polymerase. This is not comparable to Indonesia’s potential which is located on the Ring of Fire. Consequently, Indonesia holds the potential for a large geothermal ecosystem which serves as a habitat for thermophilic bacteria capable of producing thermostable DNA polymerase. The local DNA pol I gene from Geobacillus thermoleovorans Batu Kuwung, Banten, has been successfully cloned into the plasmid pET-15b and transformed into Escherichia coli K pol in isothermal amplification, making it applicable for PCR, the commercial production of GBK pol is still considered economically unfeasible. The research conducted for GBK pol still has limitations in optimization and scale-up production. Therefore, in this study optimization of expression conditions was carried out, including the speed of agitation, post-induction incubation time, working volume and scale-up production at 7,5 L bioreactor to increase efficiency, production, and expression. In this study, the optimum conditions were obtained at an agitation speed of 200 rpm, a post-induction incubation time of 4 hours and 20% working volume. Furthermore, the production of GBK pol was successfully carried out in a 7.5 L bioreactor, resulting in the highest protein concentration of 0.042 µg/µL with the optimum post-induction incubation time for 3 hours."

"Kandungan Fenol pada limbah Industri Minyak dan Gas yang melebihi baku mutu lingkungan sangat membahayakan karena fenol bersifat toksik bahkan merupakan polutan yang berbahaya menurut EPA (Environmental Protection Agency) sehingga harus di-treatment terlebih dahulu sebelum akhirnya dibuang ke lingkungan. Metode pengolahan fenol secara konvensional memiliki beberapa kekurangan sehingga digunakanlah metode biodegradasi. Pada penelitian ini, bakteri pendegradasi yang digunakan adalah Bacillus subtilis C19. Variabel yang digunakan pada penelitian ini adalah konsentrasi fenol awal, dan penambahan glukosa. Pada variasi konsentrasi fenol 10 ppm, 50 ppm, dan 100 ppm, pertumbuhan bakteri dan degradasi fenol yang paling signifikan adalah pada konsentrasi fenol 100 ppm. Pada penambahan glukosa, didapatkan glukosa memiliki sifat kompetitif terhadap fenol sehingga mempengaruhi degradasi fenol. Kinetika pertumbuhan bakteri pada berbagai konsentrasi fenol dimodelkan dengan menggunakan kinetika orde satu persamaan Monod. Kinetika laju degradasi fenol dimodelkan dengan menggunakan kinetika orde satu dan persamaan Michaelis-Menten.

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Phenol concentration in Petroleum Industry that excess is hazardous because of phenol toxicity, moreover it is one of hazardous pollutan according to EPA (Environmental Protection Agency) thus pretreatment should be conducted before wastewater is discarded into environment. Conventional phenol removal methods have some disadvantages then we use biodegradation method. In this research, bacteria that we use is Bacillus subtilis C19. The variable that we use is initial phenol concentration and glucose addition. In phenol concentration variation which is 10 ppm, 50 ppm, and 100 ppm, bacteria growth and phenol degradation are most significant in phenol 100 ppm. In glucose addition, glucose has a competitive nature towards phenol thus it can affect phenol degradation. Cell growth kinetics model in various phenol concentration use first order and Monod Equation. Degradation reaction kinetics use first order and Michaelis-Menten Equation."

"Xilitol merupakan senyawa poliol alami yang banyak ditemukan pada buah dan sayuran. Sebagai pemanis atau pengganti gula, xilitol diperbolehkan penggunaanya dalam bidang farmasi, produk kesehatan oral, dan makanan. Produksi xilitol dapat dilakukan melalui pendekatan bioteknologi; proses fermentasi. Tujuan dari penelitian ini adalah memperoleh hidrolisat tandan kosong kelapa sawit dengan konsentrasi xilosa yang tinggi, serta memperoleh kondisi optimum fermentasinya menjadi xilitol menggunakan Debaryomyces hansenii. Optimasi hidrolisis tandan kosong kelapa sawit dilakukan menggunakan katalis asam oksalat, sedangkan optimasi kondisi fermentasi produksi xilitol meliputi: konsentrasi substrat, aerasi, dan penambahan ion logam. Kondisi optimum hidrolisis dengan konsentrasi xilosa tertinggi didapatkan pada kondisi hidrolisis menggunakan 6,0 % asam oksalat dengan suhu 121°C dan tekanan 1 atm, selama 60 menit. Sedangkan dalam optimasi kondisi fermentasi, didapatkan bahwa kondisi aerasi optimum diperoleh pada keadaan semi anaerob (volume 100,0 mL dalam erlenmeyer 250,0 mL), yang menghasilkan xilitol sebesar 41,74 g/L dari konsentrasi substrat sebesar 10,0 %. Selanjutnya, dengan penambahan ion logam menyebabkan terjadi penurunan produksi xilitol. Secara berturut-turut, dihasilkan yield value sebesar 4,90 % dalam penambahan ZnSO4.7H2O, 7,58 % dalam penambahan FeSO4.7H2O, 5,59 % dalam penambahan CaCl2.2H2O, dan 6,12 % dalam penambahan CuSO4.5H2O.

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Xylitol is a polyol compound which is naturally found in many fruits and vegetables. As a sweetener or sugar substitute, xylitol is allowed for its use in pharmaceutical, oral health care product, and foods. Production of xylitol can be done through biotechnological approache; the fermentation process.

The purposes of this research is to obtain hydrolyzates of oil palm empty fruit bunch with high xylose concentration, and to obtain optimum fermentation condition into xylitol using Debaryomyces hansenii. Hydrolysis optimization of oil palm empty fruit bunch was done by using oxalic acid catalyst, while the optimization of fermentation condition for xylitol production included: substrate concentration, aeration, and metal ion addition.

The optimum condition of hydrolysis with highest xylose concentration was obtained in hydrolysis condition that used 6.0 % oxalic acid with temperature of 121°C and pressure of 1 atm, for 60 minutes. While for the optimization of fermentation conditions, it was found that the optimum aeration condition was obtained in a state of semi-anaerobic condition (100.0 mL of medium volume in erlenmeyer of 250.0 mL), the xylitol yield was 41.74 g/L from 10.0 % substrat concentration.

Furthermore, with the addition of metal ions inducing a reduction of xylitol production. Succesively, the result of its yield value are 4.90 % in addition of ZnSO4.7H2O, 7.58 % in addition of FeSO4.7H2O, 5.59 % in addition of CaCl2.2H2O, and 6.12 % in addition of CuSO4.5H2O."

"Fruktansukrase merupakan enzim ekstraselular yang biasanya diproduksi oleh Bakteri Asam Laktat (BAL). Oleh BAL, enzim ini digunakan untuk memproduksi eksopolisakarida (EPS) dari substrat sukrosa maupun substrat rafinosa. EPS memiliki potensi yang besar dalam industri farmasi, pangan dan kesehatan. Dalam penelitian sebelumnya, Fruktansukrase rekombinan diklon ke dalam Bacillus subtilis DB 403 dan dirancang untuk disekresikan keluar sel. Penelitian ini bertujuan untuk mengisolasi protein fruktansukrase rekombinan dari bakteri Bacillus subtilis dan untuk mengetahui aktivitas fruktansukrase rekombinan tersebut. Pada penelitian ini, Bacillus subtilis rekombinan ditumbuhkan dan dipanen untuk mendapatkan protein fruktansukrase di dalam supernatan kultur. Protein dieksresikan secara ekstraselular. Ke dalam supernatan lalu ditambahkan PMSF untuk mencegah terjadinya degradasi oleh protease. Selanjutnya protein diliofilisasi dengan metode freeze dry. Pelet protein diresuspensikan dalam sejumlah kecil buffer sedemikian rupa sehingga konsentrasinya pekat, setelah itu difiltrasi dengan menggunakan Amicon® Ultra-15 Centrifugal Filter Device cutoff -30 kDa untuk memisahkan fraksi protein yang berukuran besar dan kecil. Fraksi protein yang lebih besar dari 30 kDa dikumpulkan, kemudian dianalisis dengan SDS-PAGE. Sebagian fraksi tersebut dianalisis secara in situ dengan PAS-staining. Aktivitas fruktansukrase dapat diamati pada gel yang diinkubasi dengan substrat sukrosa dan substrat rafinosa berupa pita berwarna pink intensif.

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Fructansucrase is an extracellular enzyme which usually produced by Lactic Acid Bacteria (LAB). By LAB, this enzyme is used to produce exopolysaccharide (EPS) from both sucrose and rafinose substrates. EPS has huge potential in pharmaceutical, food and health industries. In previous research, fructansucrase recombinant was cloned into Bacillus subtilis DB 403 and was designed to be secreted extracelullarly. This research aims to isolate the recombinant protein fructansucrase from Bacillus subtilis and to understand the activity of this recombinant fructansucrase. Bacillus subtilis was grown and extracted to obtain the fructansucrase protein within the culture supernatant. PMSF was added into the supernatant to prevent any degradation by proteases. The supernatant was liofilized using freeze-dry method. The protein pellets were then resuspended with small volume of buffer to obtain a more concentrated sample. Subsequently, the protein was filtrated using Amicon® Ultra-15 Centrifugal Filter Device cutoff -30 kDa to separate protein filtrate by size. Protein fraction which was larger than 30 kDa was collected and analyzed by SDS PAGE. Some of the fraction was analyzed in situ using PAS-staining. Fructansucrase activity is observed on gel after incubation with both sucrose and raffinose substrate as a pink intensive band."

"Dari penelitian sebelumnya, diketahui bahwa terdapat tiga jenis bakteriosin yang disandi oleh Weissella confusa MBF8-1, yaitu Bac1, Bac2, dan Bac3. Penelitian ini bertujuan untuk menguji keberhasilan ekspresi Bac3 yang dibawa oleh Bacillus subtilis DB403 serta karakterisasinya dengan elektroforesis SDS-PAGE dan uji KHM. Keberadaan gen bac3 dikonfirmasi dengan PCR yang menunjukkan fragmen DNA berada pada 135 bp. Produksi skala besar rekombinan B. subtilis DB403 dilakukan dengan menggunakan fermenter dengan agitasi 100 rpm dan suhu 30oC. Pelet sel yang diperoleh, dipanen dengan sentrifugasi dan dipecah dengan ultrasonikasi. Pada saat pemecahan sel, PMSF sebagai penghambat protease ditambahkan ke dalam suspensi sel, kemudian disentrifugasi. Proses purifikasi menggunakan kolom HisTrap dan fraksi purifikasi diliofilisasi. Konsentrasi protein yang akan dikarakterisasi dengan elektroforesis SDS-PAGE diukur dengan menggunakan BCA Assay. Hasil elektroforesis SDS-PAGE tidak menunjukkan pita protein seperti yang diharapkan dan ini diduga karena adanya fenomena folding, hasil purifikasi menunjukkan pita berada pada ukuran 86-87 kDa. Untuk konfirmasi proses purifikasi, uji aktivitas antimikroba KHM dilakukan, hasil uji menunjukkan peptida rekombinan Bac3 memiliki aktivitas antimikroba yang lemah.

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From the previous study, it was reported that three type of bacteriocins were produced by Weissella confusa MBF8-1, they are Bac1, Bac2, and Bac3. The objectives from this study are to know the result of Bac3 expression in Bacillus subtilis DB403 and the characterization by SDS-PAGE and MIC. The existence of bac3 encoding gene was confirmed by PCR showing DNA fragment at 135 bp. Large scale production of recombinant B. subtilis DB403 is done by fermenter with the agitation was set to 100 rpm and the temperature was 30oC. The pellet cell obtained was collected by centrifugation and the cell lysed by ultrasonication. During cell lysis, protease inhibitor PMSF was added to the cell suspension, followed by centrifugation. Purification by HisTrap column was carried out and the protein was lyophilized. The concentration of protein for SDS-PAGE characterization was measured by performing BCA Assay. The SDS-PAGE did not show protein band as expected and it was assumed due to folding phenomenon problem, purification result showed at 86-87 kDa band. To confirm the purification process, antimicrobial activity assay performing MIC was carried out, the result showed weak antimicrobial activity of Bac3 recombinant peptide."

"Bakteriosin telah dikenal sebagai polipeptida kecil yang memiliki aktivitas antimikroba dan disintesis oleh banyak bakteri. Pada penelitian sebelumnya, ditemukan adanya tiga peptida dengan motif glisin ganda yang diduga bakteriosin dalam Weissella confusa MBF8-1. Ketiga bakteriosin tersebut telah berhasil diklon pada Bacillus subtilis DB403. Penelitian ini bertujuan untuk mengetahui ekspresi dan karakterisasi peptida rekombinan bakteriosin, khususnya Bac1 menggunakan metode elektroforesis Sodium Dodecyl Sulphate Polyacrilamide Gel (SDS - PAGE). Bacillus subtilis DB403 dibiakkan dan diinduksi dengan Xylosa untuk melihat ekspresi peptida rekombinan. Kemudian sel diresupensi dan dipurifikasi dengan Kolom Afinitas HisTrap FF 5 mL yang diikuti dengan liofilisasi. Karakterisasi diamati menggunakan SDS - PAGE dan dikonfirmasi menggunakan uji aktivitas antimikroba yang menunjukkan konsentrasi hambat minimal (KHM). Bakteri indikator yang digunakan adalah Leuconstoc mesenteroides TISTR120. Adanya gen bac1 dibuktikan dengan Polymerase Chain Reaction menggunakan plasmid sebagai template dan pasangan primer spesifik. Berdasarkan terjadinya misfolding dan agregasi yang disebabkan adanya penambahan Histidin tag pada bac1, peptida Bac1 tidak berhasil dikarakterisasi menggunakan SDS - PAGE. Fraksi elution buffer Bac1 menunjukkan adanya pita tunggal pada ukuran 96 kDa. Sedangkan prediksi kalkulasi berat molekul menggunakan sekuens asam amino menunjukkan bobot molekul Bac1 adalah 4,9 kDa. Hasil Uji KHM tidak menunjukkan aktivitas potensial Bac1 sebagai bakteriosin tunggal.

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Bacteriocin is a well-known ribosommally synthesized polypeptide by many bacteria, which has antimicrobial effect. In a previous study, three putative double glycine motive peptide encoded in Weissella confusa MBF8-1. These putative bacteriocins were cloned in Bacillus subtilis DB403. This study aims to observe expression and characterization recombinant bacteriocin, in particular Bac1 using Sodium Dodecyl Sulphate Polyacrilamide Gel (SDS - PAGE) method. B.subtilis DB403 was cultivated and induced with xyllose to observe the expression of recombinant peptide. Then, cell was resuspended and purified with HisTrap FF Affinity Column 5 mL, followed by lyophilization. Characterization was done by SDS ? PAGE and was confirmed by antimicrobial activity assay performing Minimum Inhibitory Concentration (MIC). Indicator bacteria used was Leuconostoc mesenteroides TISTR120. The existence of bac1 gene has been proved by Polymerase Chain Reaction (PCR) using plasmid as template and specific primer pairs. It is assumed that due to malfolding and aggreggation caused by Histidin tag added to bac1 gene, Bac1 peptide cannot be succesfully characterized by SDS - PAGE. Analysis of Bac1 fraction from elution buffer step resulted a single band at 96 kDa. While, predictive calculation of molecular weight by Bac1 amino acid sequence is 4.9 kDa. MIC assay result did not show potential activity of Bac1 as a single bacteriocin."