scholarly journals Biochemical and structural investigation of rutinosidase from Aspergillus oryzae

2020 ◽  
Author(s):  
Koki Makabe ◽  
Ruka Hirota ◽  
Yoshihito Shiono ◽  
Yoshikazu Tanaka ◽  
Takuya Koseki
Keyword(s):  
Crystal Structure ◽  
Aspergillus Oryzae ◽  
Structural Investigation ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Industrial Applications ◽  
Flavonoid Glycoside ◽  
Flavonoid Glycosides ◽  
High Catalytic Activity ◽  
Tim Barrel

The rutinosidase-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae. Biochemical and structural investigation of the purified recombinant mature AoRut, designated as rAoRutM, was performed in this study. A 1.7 Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (β/α)8 TIM barrel fold with structural similarity to rutinosidase from Aspergillus niger (AnRut) and an exo-β (1, 3)-glucanase from Candida albicans. The crystal structure revealed that the catalytic site was located in a deep cleft, similar to AnRut, and internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides, but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-β-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and the enzyme after N-deglycosylation showed a typical α-helical CD profile, however, the molar ellipticity values of the peaks at 208 nm and 212 nm varied. The Km and kcat values for the substrates modified by rutinose were higher than those for substrates modified by β-D-glucose. Importance Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in the bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases, and the other catalyzed by a diglycosidase. The present study on the biochemical and structural investigation of A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Kohei Sasamoto ◽  
Tomoki Himiyama ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Koichi Uegaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cellulose Acetate ◽  
Electron Density ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Packing ◽  
Signal Sequence ◽  
Industrial Applications ◽  
Side Chain ◽  
Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Purification and Characterization of Natural Solid-Substrate Degrading and Alcohol Producing Hyperthermostable Alkaline Amylase from Bacillus cereus (sm-sr14)

2020 ◽  
Vol 21 (9) ◽  
pp. 872-881
Author(s):  
Sumit Sahoo ◽  
Sudipta Roy ◽  
Dipannita Santra ◽  
Sayantani Maiti ◽  
Sonali Roul ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Main Property ◽  
Solid Substrate ◽  
Industrial Applications ◽  
Starch Degradation ◽  
Significant Similarity ◽  
Alcohol Production ◽  
Cazy Database ◽  
Tim Barrel ◽  
Alkaline Amylase

Objective: Amylases enzymes hydrolyze starch molecules to produce diverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1- 1, α-1-4, α-1-6, glycosidic bonds. Methods: This enzyme carrying an (α /β) 8 or TIM barrel structure is also produced containing the catalytic site residues. These groups of enzymes possess four conserved regions in their primary sequence. In the Carbohydrate-Degrading Enzyme (CAZy) database, α-amylases are classified into different Glycoside Hydrolase Families (GHF) based on their amino acid sequence. The present objective was to study one such enzyme based on its molecular characterization after purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrial applications. Results: Amylase producing bacteria Bacillus cereus sm-sr14 (Accession no. KM251578.1) was purified to homogeneity on a Seralose 6B-150 gel-matrix and gave a single peak during HPLC. MALDITOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/β hydrolase family. The enzyme showed an efficient application; favourable Km, Vmax and Kcat during the catalysis of different natural solid starch materials. Analysis for hydrolytic product showed that this enzyme can be classified as the exo-amylase asit produced a significant amount of glucose. Conclusion: Besides the purified enzyme, the present organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato and rice up to the application level in the pharmaceutical/ industrial field for alcohol production.

Development of Nanomaterials as Photo Catalysts for Environmental Applications

2020 ◽  
Vol 09 ◽  
Author(s):  
Ahmed M. Abu-Dief ◽  
W. S. Mohamed
Keyword(s):  
Water Purification ◽  
Smart Materials ◽  
Block Size ◽  
Industrial Applications ◽  
Vital Role ◽  
High Catalytic Activity ◽  
Chemical Contaminants ◽  
Environmental Threats ◽  
Photo Catalysis ◽  
Materials Used

Abstract:: Sustainability environmental lack is a growing and pivotal mater due to the issues: such as disturbances associated with biodiversity pollution, and climate change. Pollutants are the major cause of these environmental threats in the atmosphere. In recently, the nano-based photocatalyst is at the forefront of the author's interest because of its promising potential as a green chemical-based compound, high catalytic activity, the suitable and controllable surface area for wastewater treatment. Semiconductor materials in nanosized scale have electronic and optical properties depend on its building block size, which plays a vital role in developing smart materials that are well efficient for simultaneously destroying harmful chemical contaminants from our environment. This makes these materials used in many possible industrial applications such as water purification. In this Review, we report the most significant results contributing to progress in the area of environmental hazardous pollutant detection and removal focused on water purification especially through photo-catalysis to give readers an overview of the present research trends. Moreover, we analyze previous studies to indicate key principles of photo-catalysis and provide guidelines that can be used to fabricate more efficient photocatalysts.

scholarly journals Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Selfela Restu Adina ◽  
Antonius Suwanto ◽  
Anja Meryandini ◽  
Esti Puspitasari
Keyword(s):  
Fatty Acid ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Signal Sequence ◽  
Micrococcus Luteus ◽  
Acid Methyl Ester ◽  
Industrial Applications ◽  
Expression Level ◽  
Lipase Gene ◽  
Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

scholarly journals Deep learning for visualization and novelty detection in large X-ray diffraction datasets

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Lars Banko ◽  
Phillip M. Maffettone ◽  
Dennis Naujoks ◽  
Daniel Olds ◽  
Alfred Ludwig
Keyword(s):  
Thin Film ◽  
Crystal Structure ◽  
Artificial Intelligence ◽  
Deep Learning ◽  
Novelty Detection ◽  
Structural Similarity ◽  
X Ray Diffraction ◽  
X Ray ◽  
Diffraction Patterns ◽  
Post Hoc

AbstractWe apply variational autoencoders (VAE) to X-ray diffraction (XRD) data analysis on both simulated and experimental thin-film data. We show that crystal structure representations learned by a VAE reveal latent information, such as the structural similarity of textured diffraction patterns. While other artificial intelligence (AI) agents are effective at classifying XRD data into known phases, a similarly conditioned VAE is uniquely effective at knowing what it doesn’t know: it can rapidly identify data outside the distribution it was trained on, such as novel phases and mixtures. These capabilities demonstrate that a VAE is a valuable AI agent for aiding materials discovery and understanding XRD measurements both ‘on-the-fly’ and during post hoc analysis.

scholarly journals Flavonoid Glycosides in Brassica Species Respond to UV-B Depending on Exposure Time and Adaptation Time

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 494
Author(s):  
Susanne Neugart ◽  
Christiane Bumke-Vogt
Keyword(s):  
Brassica Species ◽  
Flavonoid Glycoside ◽  
Ultraviolet B ◽  
Flavonoid Glycosides ◽  
Production Cycle ◽  
Short Term ◽  
Greenhouse Production ◽  
Adaptation Time ◽  
Quercetin Glycosides ◽  
Kaempferol Glycosides

Recently, there have been efforts to use ultraviolet-B radiation (UV-B) as a biotechnological tool in greenhouses. Leafy Brassica species are mainly considered for their ability to synthesize glucosinolates and are valued as baby salads. They also have a remarkable concentration of chemically diverse flavonoid glycosides. In this study, the effect of short-term UV-B radiation at the end of the production cycle was investigated without affecting plant growth. The aim was to verify which exposure and adaptation time was suitable and needs to be further investigated to use UV as a biotechnological tool in greenhouse production of Brassica species. It is possible to modify the flavonoid glycoside profile of leafy Brassica species by increasing compounds that appear to have potentially high antioxidant activity. Exemplarily, the present experiment shows that kaempferol glycosides may be preferred over quercetin glycosides in response to UV-B in Brassica rapa ssp. chinensis, for example, whereas other species appear to prefer quercetin glycosides over kaempferol glycosides, such as Brassica oleracea var. sabellica or Brassica carinata. However, the response to short-term UV-B treatment is species-specific and conclusions on exposure and adaptation time cannot be unified but must be drawn separately for each species.

scholarly journals Discovery of Fe7O9: a new iron oxide with a complex monoclinic structure

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ryosuke Sinmyo ◽  
Elena Bykova ◽  
Sergey V. Ovsyannikov ◽  
Catherine McCammon ◽  
Ilya Kupenko ◽  
...  
Keyword(s):  
Applied Sciences ◽  
Crystal Structure ◽  
Iron Oxide ◽  
Iron Oxides ◽  
Industrial Applications ◽  
Ambient Conditions ◽  
X Ray Diffraction ◽  
X Ray ◽  
High Pressure High Temperature ◽  
Insight Into

Abstract Iron oxides are fundamentally important compounds for basic and applied sciences as well as in numerous industrial applications. In this work we report the synthesis and investigation of a new binary iron oxide with the hitherto unknown stoichiometry of Fe7O9. This new oxide was synthesized at high-pressure high-temperature (HP-HT) conditions, and its black single crystals were successfully recovered at ambient conditions. By means of single crystal X-ray diffraction we determined that Fe7O9 adopts a monoclinic C2/m lattice with the most distorted crystal structure among the binary iron oxides known to date. The synthesis of Fe7O9 opens a new portal to exotic iron-rich (M,Fe)7O9 oxides with unusual stoichiometry and distorted crystal structures. Moreover, the crystal structure and phase relations of such new iron oxide groups may provide new insight into the cycling of volatiles in the Earth’s interior.

The use of flavonoid glycosides for the identification of elm hybrids

1993 ◽  
Vol 23 (4) ◽  
pp. 611-616 ◽  
Author(s):  
Daniela Heimler ◽  
Andrea Pieroni ◽  
Lorenzo Mittempergher ◽  
Pietro Buzzini
Keyword(s):  
High Performance ◽  
Parental Species ◽  
Flavonoid Glycoside ◽  
Flavonoid Glycosides ◽  
Natural Hybrids ◽  
Probable Presence ◽  
Multivariate Discriminant ◽  
Number Of Individuals ◽  
Layer Chromatography ◽  
Putative Hybrids

The utilization of elm leaf flavonoids as biochemical markers for the identification of artificial and natural hybrids of elm species is discussed. Two to 11 individuals from controlled crosses of Ulmuscarpinifolia Gled., Ulmuspumila L., Ulmusparvifolia Jacq., and Ulmusjaponica (R.) Sarg. were examined. Five to seven individuals from each parental species, and a number of putative hybrids between U. carpinifolia and U. pumila that naturally occur in central and northern Italy, were also examined. Quantitative data on leaf flavonoid glycosides were obtained by means of high-performance thin layer chromatography and examined by multivariate discriminant analysis. The results show that it is possible to identify the hybrid obtained between these species even if the parents are unknown, provided a number of individuals of the parental species are examined; therefore, it is also possible to certify putative hybrids. The higher variability of the flavonoid glycoside data of U. carpinifolia and U. pumila and the probable presence of F2 generation individuals make the certification of natural hybrids between these two species in some cases difficult or even impossible.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

Immobilization of Eversa Lipases on Hydrophobic Supports for Production of Oleate Esters Solvent-Free

2021 ◽  
Author(s):  
Gloria Fernandez-Lorente ◽  
Daniela Remonatto ◽  
J. Vladimir Oliveira ◽  
J. Manuel Guisan ◽  
Débora Oliveira ◽  
...  
Keyword(s):  
Sunflower Oil ◽  
Low Cost ◽  
Industrial Applications ◽  
Solvent Free ◽  
Molar Ratio ◽  
High Catalytic Activity ◽  
Promising Alternative ◽  
Highly Active ◽  
Immobilized Biocatalyst ◽  
Hydrophobic Supports

Abstract Lipases are an important group of biocatalysts for many industrial applications. Two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 was immobilized on four different hydrophobic supports: Lewatit-DVB, Purolite-DVB, Sepabeads-C18, and Purolite-C18. The performance of immobilized lipases was investigated in the transesterification of sunflower oil solvent-free in an anhydrous medium. Interesting results were obtained for both lipases and the four supports, but with Sepabeads support the lipases Eversa showed high catalytic activity. However, the more stable and efficient derivative was Eversa® Transform immobilized on Sepabeads C-18. A 98 wt% of ethyl ester of fatty acid (FAEE) was obtained, in 3 hours at 40ºC, ethanol/sunflower oil molar ratio of 3:1 and a 10 wt% of the immobilized biocatalyst. After 6 reaction cycles, the immobilized biocatalyst preserved 70 wt% of activity. Both lipases immobilized in Sepabeads C-18 were highly active and stable in the presence of ethanol. The immobilization of Eversa Transform and Eversa Transform 2.0 in hydrophobic supports described in this study appears to be a promising alternative to the immobilization and application of these news lipases still unexplored.

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