Biochemical Characterization of Lipase Produced by <i>Bacillus</i>spp. Isolated from Soil and Oil Effluent

The aim of the present work was to isolate Bacillus spp. With high lipase activity;to characterize the isolates using both biochemical and molecular methods;to produce lipase using Bacillus isolates and to study the biochemical and biophysical characteristics of the produced lipase. Sixty five Bacillus isolates were isolated from soil 20 isolates from guar field soil (G), 15 isolates from Abusabein field soil (B), 15 isolates from sun flower field soil (S) and 15 isolates from oil effluent (O). Lipase producing isolates were screened;a Chromogenic plate’s method was used. Enzyme activity was quantitatively assayed. Lipase production under submerged fermentation (SMF) conditions using a production medium that contained metal salts, Tween-20 and olive oil as substrate at different period 24, 48, 72 and 96 h, the optimum pH, temperature for lipase activity was determinated and kinetics as well. The isolates showed the highest lipase activity which was identified as Bacillus sp. The optimum pH, temperature, thermostability and kinetic of the produced enzymes were found in three isolates G14, O1 and B10 with the highest enzyme activity and best stability. The isolates G14, O1 and B10 revealed the highest lipase activity of 63.4, 41.2 and 28.3 U/ml, respectively. The results showed optimum pH of the lipase activity from isolates G14, O1 and B10 8.0, 6.0 and 6.0 and the optimum temperature 40, 60 and 75˚C, respectively. Lipase enzymes from isolates O1 and B10 were found to be more thermostable after incubation time for 120 min at 90˚C. The Vmax and Km values of lipase for isolates G14, OI and B10 were 17.6, 135 and 24.4 μmole∙min−1 and 1.3, 1.6 and 0.681 mM, respectively. According to these results Bacillus spp. with high lipase activity and thermostability can be used to promote food, pharmaceuticals, paper, detergents agrochemicals industries and pollution control in Sudan.

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea （Pisum sativum L.） by sequential procedures entailing ammonium sulfate precipitation, DEAE-Sepharose column, Con-A- and Green 19-Sepharose affinity columns, hydroxyapatite column, ultra-filtration, and Sephacryl 300 gel filtration. The purified soluble acid （SAC） and alkaline （SALK） invertases had a pH optimum of 5.3 and 7.3, respectively. The temperature optimum of two invertases was 37 ℃. The effects of various concentrations of Tris-HCI, HgCI2, and CuSO4 on the activities of the two purified enzymes were examined. Tris-HCI and HgCI2 did not affect SAC activity, whereas 10 mM Tris-HCI and 0.05 mM HgCI2 inhibited SALK activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%, respectively. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM, respectively. The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa. The molecular mass of SALK was 30 kDa. Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7, respectively.

Biochemical and Immunochemical Characterization of Caenorhabditis elegans Metallothioneins I and II Induced by Cadmium

Metallothioneins in Caenorhahditis elegans (CeMT-Ⅰ and Ⅱ) were punned by the combination of gel filtration ion-exchange chromatography.and high-performance liquid chromatography.Amino acid compositions and amino-terminal sequences of CeMT-Ⅰ and Ⅱ were slightly different from those of vertebrate MTs previously reported, although cysteine residue contents were relatively high.Enzyme immunoassay using anti-CeMT-Ⅱ antibody showed the difference of antigenicity to rat MT-Ⅰ and Ⅱ and even to CeMT-Ⅰ. Immunohistochemical staining revealed the existence of CeMT-Ⅱ in the intestine and the eggs, suggesting the role of MT in detoxification and homeostasis of heavv metals. 1990 Academic Press.Inc.

Novel Protease from Aspergillus tamarii URM4634: Production and Characterization Using Inexpensive Agroindustrial Substrates by Solid-State Fermentation

This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.

Comparative Evaluation of Peptidases Produced by Penicillium corylophilum and Penicillium waksmanii in Solid State Fermentation Using Agro-industrial Residues

In this present work, the best conditions for production of peptidases under solid state fermentation by the fungi Penicillium corylophilum and Penicillium waksmanii, partial purification using Sephadex G-75 gel filtration column, as well as the biochemical characterization of the partial purified enzymes were investigated. P. corylophilum showed the best production in medium containing wheat bran, agro-industrial residue, without additives （egg albumin or casein）, in which peptidase activity reached 520 U mL^-1 and the enzyme displayed the optimum activity between pH range from 7 to 8 and 60 ℃. It also showed high stability in wide pH range and temperature until 45 ℃ for 60 min of incubation. On the other hand, P. waksmanii, the best production was noted in a medium containing wheat bran （95%） and casein （5%）, reaching 545 U mL^-1, with proteolytic optimum activity at pH 7.5 and 55 ℃. The enzyme was mainly stable in pH range from 8 to 9 and at temperatures until 45 ℃ for 60 rain of incubation. The peptidases secreted by both fungi were inhibited in the presence of phenylmethane sulfonyl fluoride, showing that they belong to the subclass of serine peptidases.

Anti-trypanosomal Activity of Bufonidae(Toad)Venom Crude Extract on Trypanosoma brucei brucei in Swiss Mice

Trypanosomiasis afflicts about 6~7 million people globally and to a large extent impedes livestock production in Africa.Naturally,trypanosomal parasites undergo genetic mutation and have developed resistance over a wide range of therapies.The utilization of animals and plants products has presented therapeutic potential for identifying novel anti-trypanosomal drugs.This study evaluated toad venom for anti-trypanosomal potency in-vivo in Swiss mice.Toads were collected from July to August 2019.The acute oral toxicity and biochemical characterization of the toad venom were determined.The experimental mice were administered various doses(130 mg/kg,173 mg/kg and 217 mg/kg)of the toad venom crude extract and 0.75 mg/mL of Diamizan Plus standard drug for the treatment of trypanosomiasis,once daily for 3 days.The in-vivo anti-trypanosomal activity was evaluated by a curative test,after infecting the mice with Trypanosoma brucei brucei.The pre-patent period was 72 hours before treatment commenced.The overall results showed that trypanosomal load was highest in the control group while the group treated with Diamizan drug had the least trypanosomal load.As such,the mean trypanosomal load in relation to treatments showed a very high significant difference(P<0.05).Also,the mean trypanosomal load in Swiss mice in relation to the highest dosage of toad venom versus Diamizan drug showed a very high significant difference(P<0.05).The mean change in relation to the haematological parameters across treatments groups varied significantly(P<0.05)with the exception of Hb which showed no significant difference(P>0.05)across treatment groups.The over 50%reduction in the trypanosomal load in the 130 mg/kg group in comparison with the control group brings to bare the anti-trypanosomal potency of the toad venom.The anti-trypanosomal activity demonstrated by the toad venom has provided basis for development of new therapeutic agents from different toad species.The study recommends further studies(both in-vivo and in-vitro)followed by the characterization of the active compounds present in the toad venom responsible for the anti-tyrpanosomal activity observed alongside the management and conservation of these species.

Expression and characterization of a CALB-type lipase from Sporisorium reillianum SRZ2 and its potential in short-chain flavor ester synthesis

A lipase from Sporisorium reilianum SRZ2(SRL)with 73%amino acid sequence identity to Candida antarctica lipase B(CALB)was cloned and overexpressed in Pichia pastoris.The recombinant SRL showed a preference for short-chain p-nitrophenyl esters.It achieved maximum activity at pH 8.0 and 65°C for p-nitrophenyl hexanoate(C6)with Km and kcar/Km values of 0.14 mmol·L-1 and 1712 min 1.mmol·L-1 at 30℃,respec-tively.SRL displayed excellent thermostability and pH stability,retaining more than 79%of its initial activity after incubation at 60℃ for 72h and 75%atpH 3to 11 for 72 h.It also maintained most of its activity in the presence of inhibitors and detergents except sodium dodecyl sulfate,and it tolerated organic solvents.SRL was covalently immobilized and successfully used for ethyl hexanoate synthesis in cyclohexane or in a solvent-free system with a high conversion yield(>95%).Furthermore,high con-version yield was also achieved for the synthesis of various short-chain flavor esters when high substrate concentra-tions of2 mol.LI were applied.This study indicated that a.CALB-type lipase from S.reiliamum SRZ2 showed great potential in organic ester synthesis.

Transcriptomic and Proteomic Analysis of Mannitol-metabolism-associated Genes in Saccharina japonica

As a carbon-storage compound and osmoprotectant in brown algae,mannitol is synthesized and then accumulated at high levels in Saccharina japonica(Sja);however,the underlying control mechanisms have not been studied.Our analysis of genomic and transcriptomic data from Sja shows that mannitol metabolism is a cyclic pathway composed of four distinct steps.A mannitol-1-phosphate dehydrogenase(M1 PDH2)and two mannitol-1-phosphatases(M1 Pase1 and MIPase2)work together or in combination to exhibit full enzymatic properties.Based on comprehensive transcriptomic data from different tissues,generations,and sexes as well as under different stress conditions,coupled with droplet digital PCR(dd PCR)and proteomic confirmation,we suggest that Sja M1 Pase1 plays a major role in mannitol biosynthesis and that the basic mannitol anabolism and the carbohydrate pool dynamics are responsible for carbon storage and anti-stress mechanism.Our proteomic data indicate that mannitol metabolism remains constant during diurnal cycle in Sja.In addition,we discover that mannitol-metabolism-associated(MMA)genes show differential expression between the multicellular filamentous(gametophyte)and large parenchymal thallus(sporophyte)generations and respond differentially to environmental stresses,such as hyposaline and hyperthermia conditions.Our results indicate that the ecophysiological significance of such differentially expressed genes may be attributable to the evolution of heteromorphic generations(filamentous and thallus)and environmental adaptation of Laminariales.

Phyllosticta species associated with citrus diseases in China

Phyllosticta species associated with diseases of four commercial Citrus species grown in China are reported.Totally,496 Phyllosticta strains were isolated from mandarins(Citrus reticulata),pomeloes(C.maxima),oranges(C.sinensis)and lemons(C.limon)in the main citrus producing regions across China,and 74 strains were selected for phylogenetic analysis.Analyses inferred from the sequences of internal transcribed spacer region(ITS1,5.8S nrDNA and ITS2),partial translation elongation factor 1-alpha(TEF1)and partial actin gene(ACT),showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known,and one undescribed species.The newly resolved taxon,Phyllosticta citrichinaensis was isolated from leaves and fruits of all four Citrus species and is introduced in this paper.This taxon caused minor damage,showing irregular spots or freckles.Phyllosticta citriasiana,associated with tan spot of pomeloes,was isolated only from pomeloes,and never from lemons,mandarins and oranges.Phyllosticta citricarpa,the citrus black spot pathogen,which is presently subjected to phytosanitary legislation in the EU and United States,was isolated from lemons,mandarins and oranges,but never from pomeloes.The isolates of P.citricarpa clustered in two subclades,one from mandarins,the other from oranges and lemons.P.capitalensis was isolated from all four Citrus species as an endophyte,causing false melanose,or together with P.citricarpa or P.citriasiana.Morphological,cultural and biochemical characters were consistent with the results of phylogenetic analysis.In addition,a specific primer pair Pca8/ITS4 was designed and selected,and its corresponding PCR procedure was developed for the detection of P.citriasiana in this study.