Produksi Enzim Fibrinolitik Tempe oleh Rhizopus oryzae FNCC 6078

ackground Rhizopus oryzae FNCC 6078 had been evaluated producing fibrinolytic enzyme under solid state fermentation. Soybean had been used to produce fibrinolytic enzyme through fermentation in tempeh. The main purpose of this study was to reveal optimum condition for fermentation. The parameters of the condition were inoculum volume, incubation period and temperature. Optimum condition was defined by maximum fibrinolytic activity. Methode Fibrinolytic activity was measured using spectrophotometry at 274 nm. Result optimum condition for producing fibrinolytic enzyme was 1,5 mL volume of inoculum of Rhizopus oryzae suspension in 25%T, 42 hours for incubation period and 35oC temperature incubation.

Gene cloning, functional expression and characterization of a novel GH46 chitosanase from Streptomyces avermitilis (SaCsn46A)

A novel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acids signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 oC and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 oC. The Km and Vmax values of this enzyme were 1.32 mg∙mL− 1, 526.32 µM∙mg− 1∙min− 1, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3 mM concentration. The enzyme can hydrolyze a variety of polysaccharides which linked by β-1,4-glycosidic bonds such as chitin, xylan and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

Biotechnology of Microorganisms Growing– Fundamentals for the Development of a Litter Biodestructor

The purpose of the research work was to select the optimal conditions for the cultivation of microorganisms. As a result of the conducted research work, the modes of growing a nitrogen-fixing culture and a microorganism with high enzymatic activity were selected and worked out. At the same time, the optimal conditions for the cultivation of Azotobacter sp were determined – the temperature optimum for cell accumulation was 30°C, for increased polysaccharide production 20 °C, aeration within 5-10 l/l/min, agitator speed-150 rpm, pH value within 6.0±0.2 units, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml. A cost-effective nutrient medium was selected for growing Pseudomonas sp. molasses-autolysate medium and optimal conditions for growing the culture: cultivation temperature 30-32 °C, aeration 1.0-1.5 l/l/ min, agitator speed 150-200 rpm, pH 6.8-7.2 units, sub-titration 5.0 % KOH, defoaming with adecanol, cultivation time-72 hours, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml.

Characterization and Phylogenetic Analysis of a Novel GH43 β-Xylosidase From Neocallimastix californiae

Degradation of lignocellulosic materials to release fermentable mono- and disaccharides is a decisive step toward a sustainable bio-based economy, thereby increasing the demand of robust and highly active lignocellulolytic enzymes. Anaerobic fungi of the phylum Neocallimastigomycota are potent biomass degraders harboring a huge variety of such enzymes. Compared to cellulose, hemicellulose degradation has received much less attention; therefore, the focus of this study has been the enzymatic xylan degradation of anaerobic fungi as these organisms produce some of the most effective known hydrolytic enzymes. We report the heterologous expression of a GH43 xylosidase, Xyl43Nc, and a GH11 endoxylanase, X11Nc, from the anaerobic fungus Neocallimastix californiae in Escherichia coli. The enzymes were identified by screening of the putative proteome. Xyl43Nc was highly active against 4-Nitrophenol-xylopyranosides with a Km of 0.72 mM, a kcat of 29.28 s−1, a temperature optimum of 32°C and a pH optimum of 6. When combined, Xyl43Nc and X11Nc released xylose from beechwood xylan and arabinoxylan from wheat. Phylogenetic analysis revealed that Xyl43Nc shares common ancestry with enzymes from Spirochaetes and groups separately from Ascomycete sequences in our phylogeny, highlighting the importance of horizontal gene transfer in the evolution of the anaerobic fungi.

Structure guided engineering of a cold active esterase expands substrate range though a stabilisation mutation that allows access to a buried water chamber

Cold active esterases represent an important class of enzymes capable of undertaking useful chemical transformations at low temperatures. EstN7 from Bacillus cohnii represents a true psychrophilic esterase with a temperature optimum below 20°C. We have recently determined the structure of EstN7 and have used this knowledge to understand substrate specificity and expands its substrate range through protein engineering. Substrate range is determined by a plug at the end of acyl binding pocket that blocks access to a buried water filled cavity, so limiting EstN7 to turnover of C2 and C4 substrates. Data mining revealed a potentially important commercial reaction, conversion of triacetin to only the 1,2-glyceryl diacetate isomer, which the EstN7 was capable of achieving. Residues M187, N211 and W206 were identified as plug residues. M187 was identified as the key plug residue but mutation to alanine destabilised the structure as whole. Another plug mutation, N211A had a stabilising effect on EstN7 and suppressed the destabilising M187A mutation. The M187A-N211A variant had the broadest substrate range, capable of hydrolysing a C8 substrate. Thus, the structure of EstN7 together with focused engineering has provided new insights into the structural stability and substrate specificity that allowed expansion of substrate range.

Isolation and detailed characterisation of the first sterigmatocystin hyperproducer mould strain in Hungary

Aspergillus strains were isolated from Hungarian mills in order to get information on the appearance of sterigmatocystin (ST) producing moulds, whose presence has never been demonstrated in Hungary. Fungal isolates were classified into nine morphotypes, sections Nigri, Nidulantes, Versicolores (two morphotypes), Circumdati, Flavi (two morphotypes), Clavati and Terrei by classical mycological assays. ST producing strains could be classified into section Versicolores. ST production of the isolates was assessed by liquid and solid phase growth experiments and compared to ST producing reference strains: Aspergillus pepii SzMC 22332, Aspergillus versicolor SzMC 22333, Aspergillus griseoaurantiacus SzMC 22334 and Aspergillus nidulans RDIT9.32. Four of our isolates marked as Km11, Km14, Km26 and Km31 showed ST production in liquid medium. ST production on solid phase corn grit substrate was measured after three weeks of incubation, and Km26 isolate proved to be the most prominent with a toxin concentration of 277.1 μg g−1, surpassing all reference strains. The toxin-producing ability of Km26 isolate was also tested in a field experiment, where corn was infected. By the end of the experiment, ST level of 19.56 μg kg−1 was measured in infected corn.Molecular taxonomic identification of the Km26 strain was performed using internal transcribed spacer (ITS), calmodulin and tubulin sequence analyses. Based on these studies, strain Km26 was identified as Aspergillus creber.Here we report that an ST-producing A. creber strain has appeared in Hungary, and the Km26 strain is the first known extreme ST-producing mould in this country. As a result of climate change, aflatoxin B1 producing Aspergillus flavus strains have appeared in Hungary in the last decade. As strain Km26 is the only A. creber isolate in Hungary so far, there is no sign of mass prevalence, and due to the lower temperature optimum of the species compared to A. flavus, its appearance is probably not related to climate change.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (DL) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that DL is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Analisis Temperatur terhadap Konversi Tepung Jagung ke Molases pada Tahap sakarifikasi

Temperature sangat mempengaruhi kosentrasi molases dalam menghidrolisis tepung jagung. Kenaikan temperature menyebabkan energy kinetic molekul meningkat. Peningkatan energi yang cukup bagi molekul reaktan akan meningkatkan laju reaksi sehingga semakin tinggi temperature konversi yang diperoleh akan semakin tinggipada temperature yang yang tidak melebihi temperature optimum enzim bekerja. Dalam menghidrolisis tepung jagung ke molases dilakukan tiga tahap yakni, gelatinase, likuifikasi, dan sakarifikasi. Metode penelitian yang digunakan yaitu metode eksperimen yang dilakukan di laboratorium. Hasil penelitian ini diperoleh nilai kadar molases, massa molases, dan nilai efisiensi. Pada temperatur (40-45) oC kadar molases yang dihasilkan yakni 8,8 %, massa molases 19,33 gr, efisiensi 64%. Pada temperatur (45-50) oC kadar molases yang dihasilkan yakni 11,6 %, massa molases 25,69 gr, dan efisiensi 86%. Pada temperatur (50-55) oC kadar molases yang dihasilkan meningkat menjadi 13 %, massa molases 26,27 gr, dan efisiensi 88%. Pada temperatur (55-60) oC kadar molases lebih meningkat menjadi 14,6 %, massa molases 28,86 gr dan efisiensi 90%.. Ini artinya semakin tinggi temperatur maka kadar molases juga akan semakin bertambah. Pada temperatur rentang (40-45) oC hanya sebagian kecil molekul yang memiliki energi aktivasi yang cukup untuk bertumbukan menghasilkan reaksi, sehingga tepung jagung yang berhasil terhidrolisis lebih sedikit, sebaliknya pada temperatur (55-60) oC energi kinetik molekul naik sehingga menyebabkan peningkatan laju reaksi. Pada temperatur tersebut adalah temperatur optimum enzim gluko amilase bekerja. Hal ini bersesuaian dengan distribusi Maxwell-Boltzmann yang menyatakan bahwa reaksi akan semakin cepat dengan adanya pertambahan temperatur.

Caulifigura coniformis gen. nov., sp. nov., a novel member of the family Planctomycetaceae isolated from a red biofilm sampled in a hydrothermal area

Pan44T, a novel strain belonging to the phylum Planctomycetes, was isolated from a red biofilm in a hydrothermal area close to the island Panarea in the Tyrrhenian Sea north of Sicily, Italy. The strain forms white colonies on solid medium and displays the following characteristics: cell division by budding, formation of rosettes, presence of matrix or fimbriae and long stalks. The cell surface has an interesting and characteristic texture made up of triangles and rectangles, which leads to a pine cone-like morphology of the strain. Strain Pan44T is mesophilic (temperature optimum 26 °C), slightly alkaliphilic (pH optimum 8.0), aerobic and heterotrophic. The strain has a genome size of 6.76 Mb with a G + C content of 63.2%. Phylogenetically, the strain is a member of the family Planctomycetaceae, order Planctomycetales, class Planctomycetia. Our analysis supports delineation of strain Pan44T from all known genera in this family, hence, we propose to assign it to a novel species within a novel genus, for which we propose the name Caulifigura coniformis gen. nov., sp. nov., represented by Pan44T (DSM 29405T = LMG 29788T) as the type strain.