CHARACTERIZATION OF PARTIALLY PURIFIED TYROSINASE ISOLATED FROM BITTER KOLA (GARCINIA KOLA)

Objective: Tyrosinase is a glycosylated, copper-containing oxidase that catalyzes the first two steps of mammalian melanogenesis as well as enzymatic browning events in damaged fruits during post-harvest handling and processing. Human skin hyperpigmentation and enzymatic browning in fruits are both undesirable. In this study, the properties and inhibitory effect of some compounds on bitter kola tyrosinase were investigated. Methods: Bitter kola tyrosinase was isolated and characterized using standard protocols. Partial purification was carried out on Sephadex G-100 loaded column chromatography. Results: Bitter kola tyrosinase was purified with a specific activity of 3.5 U/mg protein, purification fold of 2.4 and a yield of 34%. The optimum pH value was found to be 6.0 while the optimum temperature value for maximum enzyme activity was observed at 60°C. The enzyme was stable at 40oC for 20 minutes. Metals such as NaCl, KCl, MgCl2 and CaCl2 had inhibitory effect on the activity; though MgCl2 and CaCl2 had minimal effect. Also, EDTA, β-marcaptoethanol and glutathione greatly inhibited the enzyme activity at all the tested concentration. Conclusion: The properties of bitter kola tyrosinase compare very well with the tyrosinase from other sources. Also, the study was able to establish the inhibitory effect of some compounds and this could be applied in food processing industries. Peer Review History: Received: 2 November 2021; Revised: 11 December; Accepted: 25 December, Available online: 15 January 2022 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. Nazim Hussain, North East Frontier Technical University, Arunachal pradesh, India, [email protected] Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, [email protected] Prof. Dr. Ali Gamal Ahmed Al-kaf, Sana'a university, Yemen, [email protected] Similar Articles: PHYTOCHEMICAL PURIFICATION OF ACTIVE CONSTITUENTS ISOLATED FROM ROOT OF THE MEDICINAL HERB, CARALLUMA QUADRANGULA

The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, V max of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, V max of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U⋅mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Identification and partial purification of thermally stable peroxidase isoenzymes from seedlings of Vigna sp. (V) landrace Vn

Several soluble peroxidase isoenzymes are expressed in a landrace of Vigna sp. cultivated in the north of Cameroon (landrace called Vn in previous study) during seed germination. There are at least two cathodic peroxidases and eight major anodic peroxidases as shown by their electrophoretic migration at pH 7.4 under native conditions. These isoperoxidases are more expressed in roots than in shoots. They have different thermal stability, so that heat inactivation kinetics of crude peroxidase extracts from roots do not fit the first-order model. The slow and intermediate migrating groups of anodic isoperoxidases retains a substantial activity after ten minutes of incubation at 80°C and 85°C. An anodic isoperoxidase (named A6 in this study) shows in addition to this great thermal stability, a high activity in seedlings and is expressed both in roots and shoots. The combination of those characteristics makes this isoperoxidase a potential candidate for biotechnological applications. Three major anodic isoperoxidases, of which A6 and another thermostable isoperoxidase, were successfully separated from each other by ion exchange chromatography on DEAE-cellulose, after precipitation of total proteins by ice-cold acetone. This offers the prospect of being able to characterize these isoperoxidases individually in future studies.

Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum

Background and Objectives: Due to the widespread use of lipase enzymes in various industries, finding native lipase pro- ducing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed. Materials and Methods: Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in differ- ent media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20-90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads. Results: Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respec- tively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%. Conclusion: The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km of 1.47mg/ml and Vmax of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.