scholarly journals OPTIMASI PROSES UNTUK EKSPRESI GEN ENDOGLUKANASE DARI Bacillus sp. RP1 OLEH Escherichia coli BL21 (DE3)/ egc

2018 ◽  
Vol 5 (1) ◽  
pp. 44
Author(s):  
Hans Victor ◽  
Maelita Ramdani Moeis
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Process Optimization ◽  
Rice Hull ◽  
Bacillus Sp ◽  
Fermentation Time ◽  
E Coli ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Dry Cell

Process Optimization for Endoglucanase Gene Expression Derived from Bacillus sp. RP1 by Escherichia coli BL21 (DE3)/egcABSTRACTCellulases are one of the most used enzymes in industrial processes. In an effort to increase production, industries have developed strategies such as isolating new cellulase producing strains, genetic engineering and process optimization since the last 50 years. One endoglucanase producing strain, Bacillus sp. RP1 was isolated from hot springs. The ribosome binding site and coding sequence of the endoglucanase gene (egc) from Bacillus sp. RP1 was cloned into pGEM-T Easy. The recombinant plasmid was used to transform E. coli BL21 (DE3). Cloning was followed by process optimization. Medium composition was selected using Plackett-Burman design. The medium components tested were rice hull, molasses, ammonium chloride, urea and fishmeal. Rice hull and molasses were found to be the factors most influencing enzyme activity and dry cell weight, respectively. The next step involved Box-Behnken method and response surface methodology to optimize the responses against molasses concentration, rice hull concentration and fermentation time. The concentration intervals used to test were 1%, 5.5% and 10% while the fermentation time used were 24, 36 and 48 hours. The conditions which optimized both enzyme activity and dry cell weight were 7.45% molasses, 6.45% rice hull and 39.52 hours of fermentation.Keywords: Bacillus sp. RP1, E. coli BL21 (DE3), egc, Endoglucanase, optimization ABSTRAKSelulase adalah salah satu enzim yang banyak dimanfaatkan dalam berbagai industri. Sebagai upaya untuk memenuhi kebutuhan, 50 tahun terakhir dikembangkan beberapa strategi untuk meningkatkan produksi selulase yang mencakup rekayasa genetika dan optimasi proses. Karena itu, dilakukan kloning gen egc dan RBS yang berasal dari Bacillus sp. RP1 yang diisolasi dari sumber air panas ke dalam vektor pGEM-T Easy. E. coli BL21 (DE3) ditransformasikan dengan vektor yang mengandung gen egc tersebut. Setelah kloning, optimasi proses berupa desain medium turut dilakukan untuk mengoptimalkan ekspresi gen egc. Desain medium diawali dengan seleksi komposisi medium menggunakan metode Plackett-Burman. Komponen medium yang diuji adalah kulit beras, molase, amonium klorida, urea dan tepung ikan. Kulit beras dan molase diperoleh sebagai bahan yang paling berpengaruh terhadap aktivitas enzim dan berat kering sel. Tahap selanjutnya melibatkan metode statistik Box-Behnken dan metodologi respons permukaan yang bertujuan mengoptimalkan respons aktivitas enzim dan berat kering sel terhadap konsentrasi molase, konsentrasi kulit beras dan lama fermentasi. Konsentrasi yang diuji adalah 1%, 5,5% dan 10%, sedangkan lama fermentasi yang diuji adalah 24, 36 dan 48 jam. Konsentrasi optimal molase adalah 7,45% dan konsentrasi optimal kulit beras adalah 6,45% dengan lama fermentasi optimal 39,52 jam.Kata Kunci: Bacillus sp. RP1, E. coli BL21 (DE3), egc, Endoglukanase, optimasi

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

scholarly journals ISOLASI BAKTERI DARI TANAH SEBAGAI PENGHASIL SENYAWA ANTIMIKROB

Biospecies ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 46-51
Author(s):  
Sukmawati Sukmawati ◽  
FEBRIANTI ROSALINA
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Sp ◽  
E Coli

Antibiotik adalah bahan obat yang sangat memegang peranan penting dalam menanggulangi penyakit infeksi. Senyawa antimikroba dapat diperoleh dari tumbuh-tumbuhan, dan mikroba. Senyawa antimikrob yang dihasilkan oleh mikroba memiliki keunggulan dibandingkan dengan antibiotik sintetik karena memiliki sifat yang lebih efektif, sebab targetnya spesifik serta toksisitasnya rendah. Tujuan dari penelitian yang telah dilakukan ialah untuk mengisolasi bakteri dari tanah yang mampu menghasilkan senyawa antimikrob, serta untuk menguji aktivitas penghambtannya terhadap  pertumbuhan Escherichia coli dan Staphylococcus aureus. Metode yang telah digunakan dalam penelitian ini ialah isolasi, purifikasi, dan seleksi bakteri dari sampel tanah dengan metode uji penghambatan sedangkan bakteri Bacillus sp. Merupakan bakteri pembanding. Berdasarkan hasil penelitian yang diperoleh terdapat 2 isolat yang berpotensi memiliki aktivitas antimikroba yaitu isolat 1 dan isolate 4. Isolat 1 lebih berpotensi menghambat E. coli dengan indeks hambat 4.0 mm dibandingkan dengan penghambatan S. aureus dengan indeks hambat 3.1 mm. Sedangkan isolat 4 lebih berpotensi menghambat S. aureus dengan indeks hambat 2.8 mm dibandingkan dengan penghambatan terhadap E. coli dengan indeks hambat 1.4 mm.

scholarly journals Immobilization of β-Galactosidases on the Lactobacillus Cell Surface Using the Peptidoglycan-Binding Motif LysM

Catalysts ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 443 ◽  
Author(s):  
Mai-Lan Pham ◽  
Anh-Minh Tran ◽  
Suwapat Kittibunchakul ◽  
Tien-Thanh Nguyen ◽  
Geir Mathiesen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Display ◽  
Active Surface ◽  
Lactobacillus Delbrueckii ◽  
Binding Motif ◽  
Cell Weight ◽  
Lysm Domain ◽  
Dry Cell Weight ◽  
Dry Cell ◽  
Lactobacillus Spp

Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The β-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. β-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored β-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-β-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the β-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides.

scholarly journals Poly-β-Hydroxybutyrate Production by the Cyanobacterium Scytonema geitleri Bharadwaja under Varying Environmental Conditions

Biomolecules ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 198 ◽  
Author(s):  
Manoj K. Singh ◽  
Pradeep K. Rai ◽  
Anuradha Rai ◽  
Surendra Singh ◽  
Jay Shankar Singh
Keyword(s):  
Carbon Source ◽  
Growth Phase ◽  
Environmental Conditions ◽  
Carbon Sources ◽  
Stationary Growth Phase ◽  
Stationary Growth ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Phb Production ◽  
Dry Cell

The production of poly-β-hydroxybutyrate (PHB) under varying environmental conditions (pH, temperature and carbon sources) was examined in the cyanobacterium Scytonema geitleri Bharadwaja isolated from the roof-top of a building. The S. geitleri produced PHB and the production of PHB was linear with the growth of cyanobacterium. The maximum PHB production (7.12% of dry cell weight) was recorded when the cells of S. geitleri were at their stationary growth phase. The production of PHB was optimum at pH 8.5 and 30 °C, and acetate (30 mM) was the preferred carbon source.

scholarly journals Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Birgit Schilling ◽  
Nathan Basisty ◽  
David G. Christensen ◽  
Dylan Sorensen ◽  
James S. Orr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Regulatory Mechanism ◽  
Growth Conditions ◽  
Lysine Acetylation ◽  
Label Free ◽  
Content Type ◽  
E Coli ◽  
Regulatory Mode ◽  
Specific Sugar

ABSTRACT Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli. IMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

scholarly journals Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia coli

1998 ◽  
Vol 64 (3) ◽  
pp. 1018-1023 ◽  
Author(s):  
I. Tryland ◽  
L. Fiksdal
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Environmental Water ◽  
Coliform Bacteria ◽  
Galactosidase Activity ◽  
Fluorogenic Substrates ◽  
Rapid Methods ◽  
E Coli ◽  
Aerococcus Viridans

ABSTRACT Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities ofBacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified asAeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

scholarly journals Preparation of gram quantities of a purified R-factor-mediated penicillinase from Escherichia coli strain W3310

1972 ◽  
Vol 130 (1) ◽  
pp. 55-62 ◽  
Author(s):  
J. Melling ◽  
G. K. Scott
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Coli Strain ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
E Coli ◽  
R Factor

Purified penicillinase, in gram quantities, has been prepared from Escherichia coli strain W3310 by using methods developed to handle large amounts of material. The final product had a specific enzyme activity of 3.08 units/μg of protein, which was over twice as high as that reported previously (Datta & Richmond, 1966). The purified enzyme was similar to that from E. coli strain TEM, but different in molecular weight and some other respects. The differences observed may be a result of the greater purity obtained.

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