Production of β-glucosidase from Fusarium oxysporum was investigated during degradation of some cellulosic substrates (Avicel, α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production of β-glucosidase using the cellulosic substrate that supported highest yield of enzyme was examined over 192 h fermentation period and varied pH of 3.0–11.0. The β-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield of β-glucosidase (177.5 U/mg) at pH 6.0 and 30°C at 96 h of fermentation with liberation of 2.121 μmol/mL glucose. The crude enzyme had optimum activity at pH 5.0 and 70°C. The enzyme was stable over broad pH range of 4.0–7.0 with relative residual activity above 60% after 180 min of incubation. β-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity of β-glucosidase was enhanced by Mn2+ and Fe2+ with relative activities of 167.67% and 205.56%, respectively, at 5 mM and 360% and 315%, respectively, at 10 mM. The properties shown by β-glucosidase suggest suitability of the enzyme for industrial applications in the improvement of hydrolysis of cellulosic compounds into fermentable sugars that can be used in energy generation and biofuel production.
Twelve strains of Bacillus licheniformis isolated from traditionally fermented African locust bean (Parkia biglobosa) were evaluated in respect to production of protease on skim milk agar. B. licheniformis LBBL-11 exhibited the highest proteolytic activity with an average area of clear zone measuring 960 mm 2 . Production of protease from B. licheniformis LBBL-11 was further studied by growing the strain on nutrient broth. Maximum protease production was 18.4 U/mL at 48 h of growth, which coincided with the end of exponential phase. The protease from this Bacillus sp. had optimum pH of 8.0 and was stable over a wide pH range of 5.0-11.0. The optimum temperature for the protease activity was 60C. The enzyme was 95% stable at 60C after 60 min of incubation. These properties indicate possible application of B. licheniformis LBBL-11 as potential starter culture for the fermentation of African locust bean under controlled conditions of temperature and pH. PRACTICAL APPLICATIONSAfrican locust bean seeds (Parkia biglobosa) are rich in protein and usually fermented to a tasty food condiment (iru) used as flavor intensifier for soups. This is highly consumed in developing and under developed countries
Background: Proteases from bacteria are among the most important hydrolytic enzymes that have been studied due to their extracellular nature and high yield of production. Of these, alkaline proteases have potential for application in detergent, leather, food, and pharmaceutical industries. However, their usefulness in industry is limited by low activity and stability at high temperatures, extreme pH, presence of organic solvents and detergent ingredients. It is therefore very crucial to search for new alkaline proteases with novel properties from a variety of microbial sources. Results: In the present study, 21 Bacillus species isolated from organic waste sites were screened for proteolytic activity on casein agar. Bacillus brevis MWB-01 exhibited highest proteolytic activity with a clear zone diameter of 35 mm. Production of protease from B. brevis MWB-01 was investigated in optimized media after 48 h of cultivation with shaking (180 rpm) at 37°C. The protease was partially purified in a two-step procedure using ammonium sulphate precipitation and gel filtration chromatography on Sephadex G-200 column. The enzyme was purified 2.1-fold with yield of 4.6%. The purified protease had optimum temperature of 40°C with relative activity of about 50% at 50°C and was uniquely stable up to 60°C after 30 min of incubation exhibiting 63% residual activity. The enzyme had optimum pH of 8.0 and remarkably showed relative activity above 70% at pH 9.0 to 11.0 and 53% at pH 12.0, respectively and was very stable over a wide pH range (6.0 to 12.0). Ca 2+ and Mn 2+ increased protease activity with 9.8% and 3.5%, respectively; Hg 2+ and Zn 2+ strongly inhibited protease activity by 89% and 86%. The almost complete inhibition of the enzyme by phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA) confirmed the enzyme as a serine metalloprotease. The enzyme had highest compatibility with Sunlight, a commercial laundry detergent.
The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC 50 = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.
HIV‐1 protease (PR) was cocrystallized in competitive mixtures of saquinavir (SQV) and ritonavir (RTV) in an attempt to compare the relative potencies of inhibitors using a crystallographic approach. The mixture ratio of RTV/SQV was in the range of 1:1 to 50:1. The crystal form obtained with 1:1 and 5:1 ratios of RTV/SQV was monoclinic, while ratios 15:1 and 50:1 gave orthorhombic crystal form. The four crystal structures of PR/RTV/SQV were solved at 1.03, 1.12, 1.25, and 1.72 Å resolutions. The X‐ray crystal structures reveal that the crystal forms are dependent on the occupancy of either SQV or RTV in the active site of PR. At low RTV/SQV concentrations, PR/SQV complex is dominant, and at higher ratios, PR/RTV is found. The absence of a crystal structure having both inhibitors statistically disordered in the catalytic site of PR suggests that the two protein complexes are sufficiently different in properties to be discriminated in crystal growth process. The X‐ray structures of the dimeric enzyme with C2 pseudosymmetry show a 2‐fold‐disorder phenomenon for the SQV, while the RTV inhibitor is detected in a single orientation. The dominancy of PR/SQV crystal form at an equimolar mixture of inhibitor and the presence/absence of the 2‐fold disorder of inhibitors have given new insight into the relative potency of these drugs and both suggest a higher potency of SQV with respect to RTV. Research supported by Schlumberger FFTF.
Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the R-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.KEYWORDS Carbamylation, N-terminal proline, HIV-1 protease/saquinavir complex, single crystals T he HIV protease (PR) is an aspartic protease that shares sequence homology around the active site with other retroviral proteases, as the conserved Asp-ThrGly catalytic triad. 1 PR is required for proteolytic cleavage of viral Gag and Gag-Pol polyproteins into individual structural and functional proteins during viral maturation. 2 In the absence of this proteolysis, immature noninfectious virions are produced, thus making PR a prime target for structureassisted drug design in antiviral therapy. 3,4 The structure-assisted drug design and discovery process utilizes techniques such as protein crystallography, nuclear magnetic resonance (NMR), and computational biochemistry to guide synthesis of potential drugs. PR has been widely characterized biochemically and structurally, which has led to the discovery of HIV protease inhibitors (PIs). Their utilization in highly active antiretroviral therapy (HAART) has been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). 5 Recently, we have demonstrated that high-resolution X-ray crystallography can be used as a powerful method to identify the most potent PR inhibitor present in an epimeric mixture. 6 The pseudosymmetric peptidomimetic inhibitor based on a novel Phe-Pro isostere core matched the 2-fold symmetry of the homodimeric structure of the PR. Fourier maps obtained by high-resolution diffraction data (1.3 Å) clearly showed the catalytic site fully occupied by a single ordered stereoisomer. On the contrary, several X-ray crystal structures of PR complexed with more asymmetric FDAapproved inhibitors, like darunavir, nelfinavir, and saquinavir (SQV), have the catalytic site occupied by the inhibitor oriented in two almost equivalent positions related by a pseudo-2-fold symmetry.  To investigate this order/disorder phenomenon and its possible relationship with crystal packing, we have undertaken a systematic search for new crystal forms of wild-type PR in complex with SQV. Single crystals of the PR/SQV complex were obtained by a vapor diffusion technique and analyzed by X-ray diffraction. These crystals belong to a new monoclinic crystal form, which contains two dimers of the complex in the asymmetric unit. Other crystal...
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