Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate.

A novel Lip+ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip+Amy+host. Lipase hydrolyzed methyloleate into methanol that sustained PAOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip+Amy+ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy+ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip+ host to validate disparity between lipase background and PAOX1 induction in presence of methyloleate.

Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity.

Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett-Burman (PB). Of these, yeast extract, MgSO4 and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO4 (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC-MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications.

Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.

Annulation of Conjugated Azine-Imine with a Sulfoxonium Ylide in a Noncarbenoid Route to Synthesize Multisubstituted Imidazole-Fused Heterocycles.

A new [4+1]-annulation of in situ generated heterocyclic azine-aldimines with ß-keto sulfoxonium ylides has been developed. The reaction constructs N-fused imidazole rings. In the reaction, the ylides play a dual-functional role of a nucleophilic 1,1-dipolar one-carbon synthon and a source of an internal oxidant, dimethyl sulfoxide, that promotes in situ dehydrogenation to product scaffolds. The method enables access to imidazo-pyridine, pyrazine, and pyrimidine heteroaromatics.

Comprehensive Survey on Nanobiomaterials for Bone Tissue Engineering Applications.

One of the most important ideas ever produced by the application of materials science to the medical field is the notion of biomaterials. The nanostructured biomaterials play a crucial role in the development of new treatment strategies including not only the replacement of tissues and organs, but also repair and regeneration. They are designed to interact with damaged or injured tissues to induce regeneration, or as a forest for the production of laboratory tissues, so they must be micro-environmentally sensitive. The existing materials have many limitations, including impaired cell attachment, proliferation, and toxicity. Nanotechnology may open new avenues to bone tissue engineering by forming new assemblies similar in size and shape to the existing hierarchical bone structure. Organic and inorganic nanobiomaterials are increasingly used for bone tissue engineering applications because they may allow to overcome some of the current restrictions entailed by bone regeneration methods. This review covers the applications of different organic and inorganic nanobiomaterials in the field of hard tissue engineering.

Fabrication and in-vitro biocompatibility of freeze-dried CTS-nHA and CTS-nBG scaffolds for bone regeneration applications.

The thought of biodegradable organic-inorganic composites composed of natural polymer chitosan and ceramic nanoparticles (hydroxyapatite and bioglass) can be considered as a solution for hard tissue engineering. In this paper, we described a comparative assessment of chitosan-nanohydroxyapatite (CTS-nHA) and chitosan-nano-bioglass (CTS-nBG) scaffolds. The dispersion of nanoscaled hydroxyapatite (nHA) and bioglass (nBG) in chitosan remained satisfactory. The freeze-dried composite based CTS-nHA and CTS-nBG scaffolds shown porous structure. The physiochemical and morphological analysis of all samples has been performed through X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), Brunauer-Emmett-Teller (BET), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The SEM image confirmed the presence of spherically shaped nHA particles of 4.20 µm and irregularly shaped nBG particles of 6.89 µm. The TEM analysis revealed the existence of 165.52 to 255.17 nm sized nHA particles and 167.35 to 334.69 nm sized nBG particles. TEM analysis also showed the interconnected structure of CTS-nHA and CTS-nBG nanocomposites. After seven days' incubation period, the CTS-nHA and CTS-nBG scaffolds shown good mineralization behavior in simulated body fluid (SBF). The CTS-nHA scaffolds exhibited enhanced compressive strength and elastic modulus compared with the CTS-nBG sample. The cell culture experiment revealed that fabricated scaffolds had good compatibility with fibroblast cells (L929, ATCC) and MG-63 which are able to adhere, proliferate, and migrate through the porous structure. All the obtained results clearly recommend that pre-loaded hydroxyapatite and bioglass nanoparticles can enhance the apatite formation. The scaffolds with chitosan, bioglass, and hydroxyapatite have better biomechanical characteristics and allow cell growth. Therefore, these scaffolds can be perfect candidates for various hard tissue engineering applications such as bone regeneration.

High level extracellular production of recombinant γ-glutamyl transpeptidase from Bacillus licheniformis in Escherichia coli fed-batch culture.

Increasing demand of microbial γ-glutamyl transpeptidase (GGT) in food and pharmaceutical sectors raised the need for process development for high level production of the enzyme. In this respect, GGT from Bacillus licheniformis ER15 (SBLGGT) was cloned along with its native secretion signal and expressed in E. coli using different expression vectors. Native signal of the enzyme assistedits extracellular translocationin E. coli.Maximum enzyme expression was shown by construct pET51b-sblggt,in comparison to other clones, in E. coli. Shake-flask cultivation and expression using Luria-Bertani (LB) medium resulted in 2800 U/l enzyme titers in 48 h which was furtherenhancedto 4.3-fold after optimizing various cultivation conditions viz. inducer concentration, agitation, medium and induction optical density. High cell density cultivation using fed-batch fermentation strategy resulted in 20-fold increase over shake flask studies to a level of 61250 U/l. After 24 h,the specific product yield was 2355 U/g dry cell weight (DCW)with volumetric productivity of 2552 U/l/h. Of the total enzyme expressed,40% was translocated extracellularly during high cell density fed-batch fermentation resulting in an enzyme activity of 24500 U/l in the extracellular medium after 24 h. This is the highest reported enzyme titers of bacterial GGT enzyme in E. coli expression system. Thus, the current study provides a cost-effective method for the over-expression and preparation of bacterial GGT enzyme for its industrial applications.

Synthesis of Polyfunctionalized Pyrroles via a Tandem Reaction of Michael Addition and Intramolecular Cyanide-Mediated Nitrile-to-Nitrile Condensation.

A new approach for the synthesis of tetrasubstituted/functionalized NH-pyrroles from gem-diactivated acrylonitriles and TMSCN has been developed. The strategy utilizes the generation of vic-dinitrile via Michael addition and cyanide-mediated nitrile-to-nitrile cyclocondensation, which proceed in tandem guided by manifold roles of "CN". An extended application to the production of fused pyrrole has also been realized.

MTA versus Biodentine: Review of Literature with a Comparative Analysis.

An ideal dental repair material should possess certain exclusive properties such as adequate adhesive ability, insolubility, dimensional stability, biocompatibility, bioactivity etc. New materials claiming better performance are continuously being introduced in the market to optimize the care of dental patients. Biodentine has been recently introduced as the "the first all-in-one, bioactive and biocompatible material for damaged dentin replacement". Manufacturers claim that Biodentine has noticeably shorter setting time in contrast to other silicate cements such as Mineral Trioxide Aggregate (MTA) and also has better mechanical and handling properties. This article is aimed to compare the properties of MTA and Biodentine analyzing the research work done in this field so far by various researchers all across the globe.

A nitrile-stabilized ammonium ylide as a masked C-C[double bond, length as m-dash]N synthon in heterocyclization with amidine-imine: 3-component assembly to fused pyrimidine scaffolds.

A unique reactivity of a nitrile-stabilized quaternary ammonium ylide as a masked C-C[double bond, length as m-dash]N synthon in contrast to its usual sigmatropic rearrangement has been demonstrated. Its reaction with 2-aminopyridine-derived aldimine undergoes electronically-assisted nucleophilic additions and Hofmann elimination, and provides a new method to access fused pyrimidines.

Heterologous expression of γ-glutamyl transpeptidase from Bacillus atrophaeus GS-16 and its application in the synthesis of γ-d-glutamyl-l-tryptophan, a known immunomodulatory peptide.

Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16°C after 16h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5.2 fold purity. The purified enzyme had a pH optimum of 10.0 and temperature optimum of 50°C. It exhibited broad pH stability (6.0-12.0) and was thermostable (t1/2 of 54min at 50°C). The enzyme was completely inactivated by Pb2+ ions and strongly inhibited in presence of N-bromosuccinimide, azaserine and 6-diazo-5oxo-l-norleucine. Kinetic characterization of BaGGT using GpNA as a donor and glycylglycine as acceptor revealed that it had a Km of 0.15mM and 0.37mM and Vmax of 23.09µmol/mg/min and 121.95µmol/mg/min for hydrolysis and transpeptidation reactions, respectively. BaGGT also displayed broad substrate specificity for various amino acids. It was studied for its prospective use in the synthesis of an immunomodulatory peptide, γ-d-glutamyl-l-tryptophan. After optimization of various process parameters, a conversion rate of 50%, corresponding to 25mM product yield, was achieved within 6h of incubation using 50mM d-glutamine as donor and 50mM l-tryptophan as acceptor and 0.3U/mL of BaGGT in the reaction, performed at pH 10.0 and 37°C. The product was purified to homogeneity using Dowex 1×2 column and its purity was confirmed by HPLC and H1 NMR.

Comparative evaluation of the efficacy of three different irrigation devices in removal of debris from root canal at two different levels: An in vitro study.

AIM: The purpose of this in vitro study was to evaluate the cleaning efficacy of NaviTip, Max-i-Probe and Endovac in removal of debris from the root canal at 1.5 and 3.5 mm from the apex. MATERIALS AND METHODS: Forty single-rooted teeth were divided into four groups according to the root canal irrigation system (EndoVac, NaviTip, Max-i-Probe, and control). Instrumentation was done using ProFile 0.06 taper series to MAF #40. Root canals were irrigated after each file size with 1 ml of 5% NaOCl. For final irrigation 5 ml of 5% NaOCl and 17% ethylenediaminetetraacetic acid (EDTA) was used. Each group was irrigated with a different irrigation device. Four micron thick serial sections were prepared at 1.5 and 3.5 mm from the apical level and photographs were taken for the analysis. The influence of the irrigation system was evaluated using a one-way analysis of variance (ANOVA) test and unpaired t-test (P < 0.05). RESULTS: Endovac showed significantly least amount of mean percentage debris followed by Max-i-probe and NaviTip at both levels (P < 0.05). In all the experimental groups, significantly less amount of mean percentage debris was seen at 3.5 mm level than at 1.5 mm level (P < 0.05). However, the difference was statistically insignificant in case of Endovac irrigation system group. CONCLUSIONS: Amongst all the experimental groups, Endovac removed significantly more debris followed by Max-i-probe and NaviTip at both levels.