Current Biotechnology - Volume 7, Issue 4, 2018

Background: Pullulan is a neutral exopolysaccharide produced aerobically by a yeast-like fungus Aureobasidium pullulans. The distinct linkage of α (1 → 4) & α (1→ 6) in pullulan impart its nonimmunogenic, non-mutagenic and non-cancerous traits. These characteristics are being exploited in food, health care, pharmacy, lithography, cosmetics. During the last four decades, pullulan has had a breakthrough in diverse fields including gene delivery, wound healing, nano-technology, diagnostics and also in human reproductive health. Objective: The present review focused on food grade pullulan, its derivatives, and their applications. Methods: Deviations in biosynthesis and downstream processing of food grade pullulan have been reviewed in detail. Results: Pullulan can be used for a variety of applications. The food applications of pullulan powder and film have been thoroughly validated. Pullulan film, being carbon neutral, sustainable, biodegradable and palatable, provides a superior substitute to conventional petrochemical polythene films. Pullulan films could be modified to add a variety of special features. These formulations have clearly demonstrated the significance of pullulan, and its potential for more applications.

Background: β-Xylosidase plays a crucial role in xylan hydrolysis and is a necessary component in enzyme cocktails used for bioconversion of lignocellulosic biomass. The enzymatic hydrolysis of lignocellulosic biomass needs a highly balanced composition of cellulases and hemicellulases. Commercial cellulases are often poor in accessory hemicellulolytic enzymes like β-xylosidase. Hence the present investigation was focused on optimization of β-xylosidase production employing low cost technology, characterization of enzyme and its application in enzymatic saccharification of sugarcane bagasse. Methods: Newly isolated xylanolytic strain Aspergillus niger ADH-11 was exploited for production of β-xylosidase under solid state fermentation. Response surface methodology was used for optimization of β-xylosidase production under solid state fermentation. Physico chemical properties of crude β- xylosidase were evaluated. Crude enzyme was supplemented with commercial cellulases for enzymatic saccharification of mild alkali treated sugar cane bagasse. Reducing sugars from enzymatic hydrolyzate were analyzed using dinitrosalysylic acid (DNS) method and monomeric sugars were analyzed using high performance liquid chromatography (HPLC). Results: Statistical optimization by response surface methodology increased β-xylosidase production by 2.85 fold and maximum yield after optimization was 214 U/g. In addition to β-xylosidase, the crude enzyme extract was also found to have multiple xylanolytic and cellulolytic enzymes. Crude β- xylosidase was optimally active at temperature 65°C and at pH 4.0. The enzyme was found to be highly stable at its optimum temperature (65°C) up to 3 h and during storage at refrigeration temperature (5- 7°C) for 2 months. Partially purified β-xylosidase showed single activity band in zymogram. Crude β- xylosidase retained 54.52 and 84.00 % activity in the presence of xylose (12 mM) and glucose (200 mM) respectively. The synergistic action of β-xylosidase rich multiple enzymes of crude extract with commercial cellulase increased glucose, xylose and arabinose yield and also reduced cellulase dose. Conclusion: The present study established that β-xylosidase and accessory hemicellulolytic enzymes of A. niger ADH-11 have significant effect on both cellulose and xylan hydrolysis and can be used to develop a superior enzyme blend with lesser dose of commercial cellulase for saccharification of pretreated sugarcane bagasse.

Background: Peels from root and vegetable crops such as sweet potato (SP), elephant foot yam (EFY), tannia and ash gourd (AG) along with mixed vegetable wastes (MVW, comprising non-edible parts of common vegetables, rotten vegetables, seeds and pulp covering them etc.) discharged from households and restaurants constitute a major part of the biodegradable wastes. Considering the pollution hazards due to inadequate and non-judicious disposal of such wastes and the ever increasing demand for biofuel especially from the developing economies coupled with the very scanty information available on the effective valorization of such starch containing biowastes, a study was planned to tap their potential as biofuel feedstocks. Methods: As different from the typical lignocellulosic biomass, the selected processing residues had high starch content coming along with them during peeling, necessitating different pretreatment strategies. The comparative compositional and ultrastructural changes in the lignocellulo-starch biomasses (LCSBs) during two pretretaments such as simple steam (100 °C) and dilute sulfuric acid (DSA) was investigated. Two moisture regimes such as 40% and 50% moisture content(MC) and three time periods (30, 45 and 60 min.) were adopted for the steam pretreatment, while the DSA pretreatment was done at 121 °C and 0.102 MPa for two time periods (30 and 60 min.). Results: Compositional studies indicated that in addition to cellulose (11-18%) and hemicelluloses (12- 18%), LCSBs contained starch (20-32%) as a major polysaccharide (dry basis) and this could lead to differential effects during pretreatment. Lignin ranged from 7.0-10.7% in the biomasses, with the highest in ash gourd peel. Whilst steam pretreatment (40% MC; 60 min.) reduced hemicellulose, cellulose and starch by 51-53%, 27% and 25% respectively, DSA pretreatment hydrolyzed 85-94% starch, 42-48% hemicellulose and 3-15% cellulose. Delignification was more in steam (27-29%) than DSA pretreatment (10-19%). Large number of intact and deformed starch granules were seen in native biomasses and steam pretreatment resulted in gelatinization and adhesion of starch on the opened up pores. Surface morphology was altered in DSA pretreatment, with deformed/stretched pores coated with little starch. Conclusion: The study showed that starch gelatinization and clogging of pores being a major change during steam pretreatment, preferential saccharification with amylases followed by cellulases might be needed to optimize the fermentable sugar yield. However, as starch and hemicelluloses are hydrolyzed to a high extent during DSA pretreatment, the amylase and xylanase loading could be reduced at the saccharification stage.

Background: Protein kinases and phosphatases are involved in the regulation of numerous biological processes by catalyzing proteins phosphorylation and de-phosphorylation status respectively. Protein Phosphatase 2C requires divalent ion for its activity and is categorized under family protein serine/threonine phosphatases i.e. they act on serine or threonine residues. Objective: The present study is focused on genome-wide analysis of Protein phosphatase 2C genes in Glycine max (dicot) and Sorghum bicolor (monocot) using various Bioinformatics tools. Method: Tools like NCBI and SMART were used to identify the presence of PP2C genes and their catalytic domains were identified. The duplication and splicing patterns of the PP2C genes were studied with the help of Webscipio tool. Result: On the basis of conserved domains, PP2C genes were further classified into various families using multiple sequence alignment method. The presence of paralogous genes was analyzed representing a strong evolutionary relationship among these families (bootstrap value >90%). Presence of some more catalytic domains was observed along with the PP2Cc in the plants.

Background: Estrogen receptor-α (ERα) positive breast cancer is considered to be one of the most common metastatic diseases. Estrogenic signalling in breast cancer is one of the most critical oncogenic pathways. Recently Lemur Tyrosine Kinase 3 (LMTK3) was identified as a potential oncogenic ERα regulator with a significant role in endocrine resistance. Therefore, targeting LMTK3 in breast cancer would control ERα modulation and may provide a better diagnostic development and a new therapeutic target to fight these resistant and aggressive tumours. Objective: The study aimed to understand the salient structural features of LMTK3 using molecular dynamics simulation. Methods: In this computational study, we modelled 3D structure of LMTK3 domain using Iterative Threading ASSembly Refinement (I-TASSER) and studied conformational dynamics using molecular dynamics simulation. We used online computational tools and software to perform comprehensive investigation on the cavities, hydrophobicity, electrostatic potential, secondary structure topology and intra- molecular interactions in LMTK3. Results: The LMTK3 structure was observed to be stable during Molecular Dynamics (MD) simulation. We also predicted the probable binding cavities in LMTK3. In addition, we determined hydrophobic clusters and patches in LMTK3 which may be crucial for folding and stabilisation. Possible interaction sites in LMTK3 were then studied by electrostatic potential analysis based on positive and negative surfaces. From the secondary structure topology analysis, we noticed nine antiparallel β-sheets forming β- sheets topology and five hairpins were involved in forming the secondary structure. Conclusion: Our inferences from this study would be helpful in understanding the structure–function relationships of LMTK3 and also help in designing suitable inhibitors for LMTK3.

Background: The endophytes of African medicinal plants are largely underexplored despite their potential as repositories of bioactive compounds. Objective: To identify the endophytic fungi associated with Annona senegalensis and evaluate the antimicrobial and antioxidant potential of their crude ethyl acetate extracts. Method: The surface sterilization technique (using ethanol and sodium hypochlorite) was used to isolate the endophytic fungi that were identified by rDNA sequencing of the ITS region. The extracts were screened for antimicrobial activity using the agar diffusion method and evaluated for antioxidant activity using a commercial kit. The total phenolic content of the extracts was determined using the Folin- Ciocalteu method and functional groups present in the extracts were predicted using Fourier-transform infrared spectroscopy. Results: Eight fungal isolates identified as Phoma species, Epicoccum nigrum, Epicoccum sorghinum, Alternaria alternata, Alternaria tenuissima, Phaeosphaeria species and Penicillium chloroleucon were isolated from the tissues of Annona senegalensis. The extracts did not exhibit any potential antimicrobial activity and the extract obtained from Epicoccum sorghinum demonstrated both the highest total phenolic content (28.85±1.14 mg GAE/g dry weight) and total antioxidant capacity (593.46 ± 1.86 μM CRE). A strong positive linear correlation (r = 0.9556) was found between antioxidant capacity and phenolic content. The Fourier- transform infrared spectral analysis of the crude extracts from Epicoccum sorghinum confirmed the presence of molecules carrying bonded hydroxyl functional groups characteristic of phenolic compounds. Conclusion: The preliminary results indicate that the isolated fungal endophytes from Annona senegalensis belong to the phylum Ascomycota and have potential as sources of natural antioxidants.

Background: Medicinal plants play an important role in the search for bioactive strains of endophytic fungi. Objective: The aim of the study was to isolate and identify the endophytic fungi associated with Kigelia africana and Warburgia salutaris and evaluate their bioactivity. Methods: Endophytic fungi were isolated from the stem and leaf tissues of Kigelia africana and Warburgia salutaris and identified by rDNA sequencing of the ITS region. Crude extracts were screened for antimicrobial activity using the agar diffusion method and evaluated for total antioxidant activity using a commercial kit. The metabolites present in the extracts were predicted using Gas Chromatography-Mass Spectrometry (GC-MS). Results: Eleven endophytic fungi identified as Penicillium chloroleucon, Alternaria solani, Coprinellus micaceus, Cryptococcus cf. taibaiensis, Vishniacozyma victoriae, Alternaria alternata and Cystobasidium larynges were isolated from Warburgia salutaris. Seven more endophytic fungi identified as Penicillium chloroleucon, Myrothecium gramineum, Phomopsis sp., Alternaria brassicae, Cercospora chrysanthemi, Cladosporium uredinicola, and Aureobasidium leucospermi were isolated from Kigelia africana. The isolate Cladosporium uredinicola from Kigelia africana was shown to have both antioxidant (175.42±2.53 μM copper reducing equivalents) and antimicrobial activity (against E. coli ATCC1056). The main constituents in the crude ethyl acetate extract of the isolate Vishniacozyma victoriae from Warburgia salutaris were identified as N- hydroxymethylacetamide (33.14%), 26- hydroxycholesterol (8.21%), formic acid, butyl ester (1.14%), cyclotetracosane (0.70%), 1-heptacosanol (0.66%), benzo [h] quinolone, 2, 4-dimethyl (0.57%), and pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro- 3-(9-phenylmethyl) – (0.50%) through GC-MS spectra analysis. Conclusion: The isolate Cladosporium uredinicola has potential as a source of antimicrobial and antioxidant bioactive compounds and the isolate Vishniacozyma victoriae produces a diverse range of metabolites.

Background: Whey is a watery part (serum) that remains after the coagulation of milk by acid or proteolytic enzymes and it contains mainly, lactose, proteins, minerals and water. Most of the countries have large dairy and cheese production units which generate huge amounts of whey per liter of milk processed. However, land disposal of whey as a waste product has been widely practiced all over the world that poses serious pollution problems. Thus, it is highly essential that whey has to be processed further to generate more valuable products and subsequently to make it suitable for safe disposal. The aim of the present study was to develop an economical method, so the dairy industry may adapt it to process whey to make it suitable for safe disposal. Methods: The membrane chromatography was used to develop an economical method for the separation of whey proteins. Cellulose fiber treated with chlorosulfonic acid and commercially available adsorber Sartobind® S was used to optimize the separation of whey protein isolate. Results: Dead end filtration study showed 1.02 mg / cm2 qm (maximum binding capacity) for cellulose fiber treated with chlorosulfonic acid and 1.3 mg / cm2 qm for Sartobind® S. Cellulose fiber showed 2.7% decrease in adsorption at 30th cycle whereas 26% decrease in adsorption was obtained at 8th cycle for Sartobind® S. The 19.0 fold higher surface area and 200 times higher feed volume was used for cross flow filtration study. It has showed 1.79 mg / cm2 qm for cellulose fiber and 3.2 mg / cm2 qm for Sartobind® S. The average flux was 543 ± 36 l / m2 / h for cellulose fiber and it was 43 ± 0.0 l / m2 / h for Sartobind® S. Electrophoresis of sample eluted at pH 7.0 showed three bands of major whey proteins for cellulose fiber whereas two bands for Sartobind® S. Cellulose fiber showed high potential for industrial applications. Conclusion: In comparison to commercially available adsorber such as Sartobind® S, which has showed higher whey protein binding capacity (qm). The cellulose fiber treated with CSA appears to be more suitable for industrial applications. As it has shown higher flux (543 ± 36 l / m2 / h) and high flux regeneration capacity with minimum washings that ensures its longer life.