Sustainable green approach to synthesize Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Syzygium cumini and its application in functional stability of microbial cellulases

Synthesis of nanomaterials following green routes have drawn much attention in recent years due to the low cost, easy and eco-friendly approaches involved therein. Therefore, the current study is focused towards the synthesis of Fe3O4/α-Fe2O3 nanocomposite using waste pulp of Jamun (Syzygium cumini) and iron nitrate as the precursor of iron in an eco-friendly way. The synthesized Fe3O4/α-Fe2O3 nanocomposite has been extensively characterized through numerous techniques to explore the physicochemical properties, including X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, Ultraviolet-Vis spectroscopy, field emission scanning electron microscope, high resolution transmission electron microscope and vibrating sample magnetometer. Further, efficiency of the Fe3O4/α-Fe2O3 nanocomposite has been evaluated to improve the incubation temperature, thermal/pH stability of the crude cellulase enzymes obtained from the lab isolate fungal strain Cladosporium cladosporioides NS2 via solid state fermentation. It is found that the presence of 0.5% Fe3O4/α-Fe2O3 nanocomposite showed optimum incubation temperature and thermal stability in the long temperature range of 50–60 °C for 15 h along with improved pH stability in the range of pH 3.5–6.0. The presented study may have potential application in bioconversion of waste biomass at high temperature and broad pH range.

Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Preparation and in vivo toxicity study of allantoin incorporated hyaluronic acid-L-cysteine oral solution: A future treatment for mucositis

Background: Thiolated hyaluronic acid (HA) with interesting properties, such as muco-adhesiveness, enzyme inhibitory, permeation enhancing, and release controlling properties can be applied for drug delivery in various diseases like mucositis. The purpose of this study was to evaluate the stability and toxicity of thiol modified HA by the aid of L-cysteine ethyl ester hydrochloride (Cys) named (HA-Cys) and allantoin (Alla) incorporated HA-Cys (HA-Cys-Alla) to reveal their potential for the future treatment of mucositis. Methods: The HA modification and drug incorporation were investigated using FTIR spectroscopy. The evaluation of in vitro cytotoxicity on Caco-2 cell line by means of MTT assay and in vivo toxicity by measuring the hematological and biochemical parameters in rats were performed. The appearance stability of HA-Cys and HA-Cys-Alla was evaluated at room and refrigerator temperatures over time. In addition, the stability of HA-Cys and HA-Cys-Alla subjected to heating and cooling, freeze-thaw, centrifugal forces, as well as the pH stability under the above-mentioned conditions were also investigated. Results: The results indicated that the synthesized HA-Cys and HA-Cys-Alla with pseudo-plastic rheological behavior demonstrated excellent stability at refrigerator temperature. Although HA-Cys showed good stability, the HA-Cys-Alla revealed color change at room temperature. Moreover, despite no much resistance of HA-Cys and HA-Cys-Alla against the heating-cooling test, the samples exhibited good resistance against freeze-thaw and centrifugal forces. Also, convenient pH stability and high in vitro and in vivo biocompatibility were observed. Conclusions: The low in vitro and in vivo toxicity and convenient stability of HA-Cys-Alla has introduced it as a proper candidate for future clinical applications.

A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L−1 and kcat/Km 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Purification and Characterization of a Thermo- and pH-Stable Laccase From the Litter-Decomposing Fungus Gymnopus luxurians and Laccase Mediator Systems for Dye Decolorization

An extracellular laccase (GLL) was purified from fermentation broth of the litter-decomposing fungus Gymnopus luxurians by four chromatography steps, which resulted in a high specific activity of 118.82 U/mg, purification fold of 41.22, and recovery rate of 42.05%. It is a monomeric protein with a molecular weight of 64 kDa and N-terminal amino acid sequence of AIGPV TDLHI, suggesting that GLL is a typical fungal laccase. GLL demonstrated an optimum temperature range of 55°C–65°C and an optimum pH 2.2 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It displayed considerably high thermostability and pH stability with about 63% activity retained after 24 h at 50°C, and 86% activity retained after 24 h at pH 2.2, respectively. GLL was significantly enhanced in the presence of K+, Na+, and Mg2+ ions. It demonstrated Km of 539 μM and kcat/Km of 140 mM–1⋅s–1 toward ABTS at pH 2.2 and 37°C. Acetosyringone (AS) and syringaldehyde (SA) were the optimal mediators of GLL (0.4 U/ml) for dye decolorization with decolorization rates of about 60%–90% toward 11 of the 14 synthetic dyes. The optimum reaction conditions were determined to be mediator concentration of 0.1 mM, temperature range of 25°C –60°C, and pH 4.0. The purified laccase was the first laccase isolated from genus Gymnopus with high thermostability, pH stability, and effective decolorization toward dyes, suggesting that it has potentials for textile and environmental applications.

Influence of the Chelation Process on the Stability of Organic Trace Mineral Supplements Used in Animal Nutrition

The effect of the chelation process on the pH-dependent stability of organic trace minerals (OTMs) used as mineral supplements in animal nutrition was assessed using analytical techniques such as potentiometry, Fourier Transform Infrared Spectroscopy (FTIRS) and amino acid profiling. The aim was to understand the influence and relative importance of the manufacturing conditions on mineral chelation and the subsequent pH stability of OTMs. A selection of OTMs were assessed over a wide pH range to account for the typical environmental changes encountered in the gastrointestinal (GI) tract. In the case of proteinate type products, the potentiometric assessment of free mineral concentration indicated that the hydrolysis procedure used to generate the chelating peptides was the major influencer of the pH stability of the products. Many products are available under the umbrella term “OTMs”, including amino acid complexes, amino acid chelates, polysaccharide complexes and proteinates. Significant differences in the pH-dependent stability of a range of commercially available OTMs were observed.

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Aminooligosaccharides possess various biological activities and can exploit wide applications in food, pharmaceutical and cosmetic industries. Commercial aminooligosaccharides are often prepared by the hydrolysis of chitin and chitosan. In this study, a novel GH family 20 β-N-acetylhexosaminidases gene named AoNagase was cloned from Aspergillus oryzae and expressed in Pichia pastoris. The purified AoNagase had maximal activity at pH 5.5 and 65°C. It exhibited good pH stability in the range of pH 6.0–7.5 and at temperatures below 50°C. AoNagase was capable of hydrolyzing not only colloidal chitosan (508.26 U/mg) but also chitin (29.78 U/mg). The kinetic parameters (Km and Vmax) of AoNagase were 1.51 mM, 1106.02 U/mg for chitosan and 0.41 mM, 40.31 U/mg for colloidal chitin. To our knowledge, AoNagase is the first GH family 20 β-N-acetylhexosaminidase capable of hydrolyzing both chitosan and chitin. AoNagase is an endo-type β-N-acetylhexosaminidases and can potentially be used for the manufacturing of aminooligosaccharides.