Current Biotechnology - Volume 8, Issue 1, 2019

Background: Laccases are important enzymes that have numerous applications in different biotechnological sectors. Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on coconut fiber and characterize different physical and kinetic properties under both free and immobilize conditions. Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through crosslinking by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH, thermostability, the effect of additives, activation energy, Km and Vmax for free and immobilized laccase were also analyzed. Recycling stability of the immobilized laccase was further determined. Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy (Ea) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol-1 respectively. Immobilized laccase showed higher melting temperature (Tm) of (82.5°C) than free enzyme (73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobilized condition laccase retained #131;80% of its initial activity after use till six repeated cycles. Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective process for different biotechnological and industrial applications.

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Methods: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

Background: Musa paradisiaca (Banana plant), which belongs to the family of Musaceae, is a well-known herbaceous flowering edible plant. The flower, fruit, and stem part of the plant have been used for nutrients and health benefits. Objective: The aim of this study is to determine the secondary metabolites, proximate composition, minerals, heavy metals, and anti-oxidant activity of three edible parts (flower, unripe fruit, and stem) of Musa paradisiaca. Methods: The content of alkaloid and tannin was determined by simple titrimetric method and colorimetric method was used for the determination of the content of phenol and flavonoid. Association of Official Analytical Chemicals (AOAC) method was used for the determination of proximate composition and the content of trace elements was analyzed by using atomic absorption spectrophotometer. The anti-oxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and hydrogen peroxide (H2O2) radical scavenging assay. Results: The results indicated that three edible parts (flower, unripe fruit, and stem) of the banana plant contained a good amount of secondary metabolites (such as alkaloid, phenol, flavonoid, and tannin) and also primary metabolites (such as carbohydrate, protein, and fat). Banana fruit contained a high amount of energy (261.31 kcal/100g) compared to the other two parts (flower, and stem). Iron, potassium, phosphorus, calcium were present in these three edible parts of the banana plant. Lead was found in negligible amount and arsenic was not detected. Fifty percent of ethanolic extract of three edible parts of the banana plant showed significant DPPH free radical scavenging and H202 radical scavenging activity as compared to standard ascorbic acid. Conclusion: Based on these findings, three edible parts of Musa paradisiaca may be recommended as a good source of nutrients.

Background: Plant in vitro culture systems serve as a useful tool to study the regulatory routes which are related to plant growth and survival under altered environmental conditions. Methods: Callus culture of Suaeda monoica and Suaeda nudiflora were established for studying the salt tolerance mechanism at the cellular level. Calli of both the species were induced from seedling’s epicotyls on Murashige and Skoog (MS) medium supplemented with a different combination of auxin and cytokinins. A sequential stress treatment was given to the callus of both the species. The growth rate of callus, osmolytes and antioxidant activities was investigated after 28 days. A control callus was maintained in each experiment without any salt in the growth medium. Results: Efficient callus regeneration was obtained by exposing the callus tissue to MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/l), benzylaminopurine (BAP, 0.5 mg/l) and 2,4-D (0.5 mg/l), kinetin (Kn, 0.25 mg/l) for S. monoica and S. nudiflora, respectively. A substantial increase was observed in proline content and a strong positive correlation was found between the total phenolic content and antioxidant activity under increasing salt concentrations. Conclusion: This is the first report on S. monoica callus regeneration. The specific cell lines which were generated through callus culture under sequential saline conditions provide a promising foundation for studying salinity induced expression of enzymes. Further comparison of transcriptomic profiles of control and salt-treated callus cultures can serve as a promising system for the detection of genes responsible for the change in expression under salt stress.

Background: The two wild plants viz. Sphenoclea zeylanica and Sphaerantus peguensis are seasonally consumed as vegetables by the Bodo people in Assam of North East India. Wild vegetables are considered as one of the cheapest sources for human nutrition that contains rich sources of numerous minerals and bioactive compounds which on consumption can contribute several health benefits against various diseases. Objective: The aim of the present study is to investigate amino acid profiles, antimicrobial property and anti-nutritional contents of the two wild edible plants. Methods: Amino acid profiles were determined by using ultra-performance liquid chromatography, antimicrobial activities of aqueous and methanol extracts of the plants were tested following the disc diffusion method against Bacillus cereus, Staphylococcus aureus, Proteus vulgaris and Escherichia coli, and anti-nutritional contents were evaluated based on the reported methods. Results: The total amino acid content found in S. zeylanica was 42.87 mg/g dry weight and it was found to be 32.65 mg/g dry weight in S. peguensis. The methanol extracts of the plants are exhibiting antibacterial activities against all the studied microorganisms. However, aqueous extracts showed no antibacterial activity against P. vulgaris and B. cereus. In this study, S. zeylanica species showed higher levels of anti-nutritional contents compared to S. peguensis. Conclusion: In the study, higher levels of essential amino acids were detected in S. zeylanica compared to S. peguensis. The methanol extracts of the plants showed more effective antimicrobial activities in comparison to the aqueous extracts and this may be due to the presence of antimicrobial compounds which are more readily soluble in methanol.