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 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 Microbioreactor for lower cost and faster optimisation of protein production

The Analyst ◽  
2020 ◽  
Vol 145 (18) ◽  
pp. 6148-6161 ◽  
Author(s):  
Mayur Parekh ◽  
AbdulAziz Ali ◽  
Zulfiqur Ali ◽  
Simon Bateson ◽  
Fathi Abugchem ◽  
...  
Keyword(s):  
Dissolved Oxygen ◽  
Protein Production ◽  
Lower Cost ◽  
Cell Weight ◽  
Growth Profile ◽  
Dry Cell Weight ◽  
Dry Cell

Microbioreactor system, with inset the microbioreactor element, and an example cultivation growth profile showing dissolved oxygen, pH and dry cell weight.

scholarly journals Fast media optimization for mixotrophic cultivation of Chlorella vulgaris

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Valerie C. A. Ward ◽  
Lars Rehmann
Keyword(s):  
Cell Density ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Accumulation Rate ◽  
Lipid Production ◽  
Total Lipid Content ◽  
Mixotrophic Cultivation ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Dry Cell

AbstractMicroalgae can accumulate large proportions of their dry cell weight as storage lipids when grown under appropriate nutrient limiting conditions. While a high ratio of carbon to nitrogen is often cited as the primary mode of triggering lipid accumulation in microalgae, fast optimization strategies to increase lipid production for mixotrophic cultivation have been difficult to developed due to the low cell densities of algal cultures, and consequently the limited amount of biomass available for compositional analysis. Response surface methodologies provide a power tool for assessing complex relationships such as the interaction between the carbon source and nitrogen source. A 15 run Box-Behnken design performed in shaker flasks was effective in studying the effect of carbon, nitrogen, and magnesium on the growth rate, maximum cell density, lipid accumulation rate, and glucose consumption rate. Using end-point dry cell weight and total lipid content as assessed by direct transesterification to FAME, numerical optimization resulted in a significant increase in lipid content from 18.5 ± 0.76% to 37.6 ± 0.12% and a cell density of 5.3 ± 0.1 g/L to 6.1 ± 0.1 g/L between the centre point of the design and the optimized culture conditions. The presented optimization process required less than 2 weeks to complete, was simple, and resulted in an overall lipid productivity of 383 mg/L·d.

scholarly journals The Effect of Mixed Wastewaters on the Biomass Production and Biochemical Content of Microalgae

Energies ◽  
2019 ◽  
Vol 12 (18) ◽  
pp. 3431 ◽  
Author(s):  
Park ◽  
Ahn ◽  
Park ◽  
Ji ◽  
Choi
Keyword(s):  
Acid Mine Drainage ◽  
Biomass Production ◽  
Mine Drainage ◽  
Basal Medium ◽  
Biofuel Production ◽  
Piggery Wastewater ◽  
Cell Weight ◽  
Acid Mine ◽  
Dry Cell Weight ◽  
Dry Cell

The effect of ammonia and iron concentration in Bold Basal Medium and mixed wastewater (including pretreated piggery wastewater and acid mine drainage) on biomass production and biochemical content (lipid and ß-carotene) of microalgae (Uronema sp. KGE 3) was investigated. Addition of iron to the Bold Basal Medium enhanced the growth, lipid, and ß-carotene of Uronema sp. KGE 3. The highest dry cell weight, lipid content, and lipid productivity of KGE 3 were 0.551 g L-1, 46% and 0.249 g L-1 d-1, respectively, at 15 mg L-1 of Fe. The highest ß-carotene was obtained at 30 mg L-1 of Fe. The biomass production of KGE 3 was ranged between 0.18 to 0.37 g L-1. The microalgal growth was significantly improved by addition of acid mine drainage to pretreated piggery wastewater by membrane. The highest dry cell weight of 0.51 g L-1 was obtained at 1:9 of pretreated piggery wastewater by membrane and acid mine drainage for KGE 3. The removal efficiencies of total nitrogen and total phosphate was ranged from 20 to 100%. The highest lipid and ß-carotene content was found to be 1:9. Application of this system to wastewater treatment plant could provide cost effective technology for the microalgae-based industries and biofuel production field, and also provide the recycling way for pretreated piggery wastewater and acid mine drainage.

scholarly journals Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

2003 ◽  
Vol 185 (12) ◽  
pp. 3485-3490 ◽  
Author(s):  
Teruyuki Kobayashi ◽  
Mari Shiraki ◽  
Tomoko Abe ◽  
Akinori Sugiyama ◽  
Terumi Saito
Keyword(s):  
Stationary Phase ◽  
Inclusion Bodies ◽  
Ralstonia Eutropha ◽  
Monomer Unit ◽  
Null Mutant ◽  
Soluble Enzyme ◽  
Cell Weight ◽  
Purification And Properties ◽  
Dry Cell Weight ◽  
Dry Cell

ABSTRACT An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2Reu) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2 Reu . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1Reu). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.

scholarly journals THE KINETICS BOTH OF GROWTH AND METABOLITE PRODUCTION OF X.CAMPESTRIS USING OF 4% LIQUID SUGAR SUBSTRATE FROM CASSAVA HYDROLISATE

2017 ◽  
Vol 6 (1) ◽  
pp. 45-49
Author(s):  
Nancy Siti Djenar ◽  
Edi Wahyu Sri Mulyono
Keyword(s):  
Growth Rate ◽  
Xanthan Gum ◽  
Maximum Growth ◽  
Process Conditions ◽  
Metabolite Production ◽  
Method Of Calculation ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Kinetics Of ◽  
Dry Cell

X. campestris is aerobic bacteria producing extracellular biopolymers (EPS, extracellular polysaccharide) known as xanthan gum. To determine the technology and the process conditions appropriate to the formation of this product, therefore the understanding of both the kinetics of growth and metabolite production of bacteria is needed. In this research, to assess the growth of X. campestris using the method of calculation of dry cell weight. For determining the kinetics of production of metabolite used substrates of 4% liquid sugar from cassava starch hydrolysate. From this research was showed that X. campestris maximum growth in NB medium obtained in about 58thhour, at the growth rate of about 0.04 g / hour, stationary phase obtained at the 60th hour with a maximum dry cell weight of 2.7688 g/L and specific growth rate (?) of X. campestris amounted to 0.043 hour-1. Based on the kinetic curves both on growth and its metabolite production, X.campestris has non-growth associated product pattern. In this case the production of xanthan gum occured after cell growth stopped then its product is a secondary metabolite with highest amount of 3.73 g / L at 102nd hour, ie the 4th day of fermentation. Overall of this research indicated that Nutrient Broth (NB) may be used for the growth of X. campestris. But based on the value of ? above, the rate of cell reproduction was still low. Liquid sugar can be used as a substrate to produce xanthan gum. However to increase its productivity, there should be an addition of other carbon or energy and nitrogen sources.

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