Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

BackgroundWe aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation.ResultsThe results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation.ConclusionsThese findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.     

Biochemical characterization of three Aspergillus niger β-galactosidases

Backgroundβ-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of β-galactosidase, but the properties of this enzyme are incompletely studied.ResultsThree new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative β-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal β-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4–5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All β-galactosidases performed transgalactosylation activity optimally in an acidic environment.ConclusionsThree new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three β-galactosidase family members with remarkably different biochemical properties.     

Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

BackgroundThe main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size.ResultsThe isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase.ConclusionsThis is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.How to cite: Liu S, Ahmed S, Zhang C, et al. Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.003     

Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein

BackgroundBiologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production.ResultsHalophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma.ConclusionThe use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.How to cite: Delgado-García M, Flores-Gallegos AC, Kirchmayr M, et al. Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.03.001.     

Oxidative foliar photo-necrosis produced by the bacteria Pseudomonas cedrina

BackgroundAlthough bioactive metabolites capable of causing oxidative photo-necrosis in plant tissues have been identified in fungi, little is known about this type of mechanism in bacteria. These metabolites act as photosensitizers that generate reactive oxygen species (ROS) capable of causing damage to cells. In addition, these metabolites can pass into an energetically excited state when they receive some luminous stimulus, a condition in which they interact with other molecules present in the environment, such as molecular oxygen (O₂), also known as triplet oxygen (³O₂), generating ROS.ResultsThe suspension of the bacterial culture of Pseudomonas cedrina was shown to produce foliar necrosis in papaya leaves (Carica papaya L.) only in the presence of sunlight, which is evidence of photosensitizing mechanisms that generate singlet oxygen (¹O₂). From the chemical study of extracts obtained from this bacteria, 3-(4-(2-carboxipropyl) phenyl) but-2-enoic acid (1) was isolated. This compound, in the presence of light and triplet oxygen (³O₂), was able to oxidize ergosterol to its peroxide, since it acted as a photosensitizer producing ¹O₂, with which it was corroborated that a photosensitization reaction occurs, mechanism by which this bacterium could prove to cause oxidative foliar photo-necrosis.ConclusionsP. cedrina was able to induce oxidative foliar photo-necrosis because of its potential ability to produce photosensitizing metabolites that generate singlet oxygen in the plants it colonizes. Based on the above, it can be proposed that some bacteria can cause oxidative foliar photo-necrosis as an important mechanism in the pathogenesis of host species.How to cite: Mendoza G, Sánchez-Tafolla L, Trigos A. Oxidative foliar photo-necrosis produced by the bacteria Pseudomonas cedrina. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.007     

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake,and investigations into its prebiotic activity

BackgroundManno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain.ResultsGalactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose.ConclusionsThe effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.How to cite: Zhang R, Li X-Y, Cen X-L, et al. Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.11.001     

Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail

BackgroundCurrent commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields.ResultsTo improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran β-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced.ConclusionsOur method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.     

Enhancement of ethanol production efficiency in repeated-batch fermentation from sweet sorghum stem juice: Effect of initial sugar,nitrogen and aeration

BackgroundEthanol concentration (P_E), ethanol productivity (Q_P) and sugar consumption (SC) are important values in industrial ethanol production. In this study, initial sugar and nitrogen (urea) concentrations in sweet sorghum stem juice (SSJ) were optimized for high P_E (≥ 10%, v/v), Q_P, (≥ 2.5 g/L·h) and SC (≥ 90%) by Saccharomyces cerevisiae SSJKKU01. Then, repeated-batch fermentations under normal gravity (NG) and high gravity (HG) conditions were studied.ResultsThe initial sugar at 208 g/L and urea at 2.75 g/L were the optimum values to meet the criteria. At the initial yeast cell concentration of ~ 1 × 10⁸ cells/mL, the P_E, Q_P and SC were 97.06 g/L, 3.24 g/L·h and 95.43%, respectively. Repeated-batch fermentations showed that the ethanol production efficiency of eight successive cycles with and without aeration were not significantly different when the initial sugar of cycles 2 to 8 was under NG conditions (~ 140 g/L). Positive effects of aeration were observed when the initial sugar from cycle 2 was under HG conditions (180–200 g/L). The P_E and Q_P under no aeration were consecutively lower from cycle 1 to cycle 6. Additionally, aeration affected ergosterol formation in yeast cell membrane at high ethanol concentrations, whereas trehalose content under all conditions was not different.ConclusionInitial sugar, sufficient nitrogen and appropriated aeration are necessary for promoting yeast growth and ethanol fermentation. The SSJ was successfully used as an ethanol production medium for a high level of ethanol production. Aeration was not essential for repeated-batch fermentation under NG conditions, but it was beneficial under HG conditions.How to cite: Sriputorn B, Laopaiboon P, Phukoetphim N, et al. Enhancement of ethanol production efficiency in repeated-batch fermentation from sweet sorghum stem juice: Effect of initial sugar, nitrogen and aeration. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.06.001     

Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts

BackgroundMicrobial oils produced by diverse microorganisms are being considered as alternative sources of triglycerides for biodiesel production. However, the standalone production of biodiesel from microorganisms is not currently economically feasible. In case of yeasts, the use of low-value nutrient sources in microbial production and the implementation of cost-efficient downstream processes could reduce costs and make microbial lipids competitive with other commodity-type oils in biodiesel production. Industrial biodiesel synthesis from oleaginous seeds is currently based on a multistep process. However, a simple process called in situ transesterification (ISTE), which takes place within the biomass without a previous lipid extraction step, is receiving increasing interest. In this work, the optimal conditions for an ISTE process to obtain biodiesel from previously selected oleaginous yeast (Rhodotorula graminis S1/S2) were defined using the response surface methodology (RSM).ResultsUsing the RSM approach, the optimal conditions for the maximum yield with minimum reaction time included a methanol-to-biomass ratio of 60:1, 0.4 M H₂SO₄, and incubation at 70°C for 3 h. The optimized in situ process yield was significantly higher (123%) than that obtained with a two-step method in which fatty acids from saponifiable lipids were first extracted and then esterified with methanol. The composition of the fatty acid methyl ester mixture obtained from R. graminis S1/S2 by ISTE met Uruguayan standards for biodiesel.ConclusionThe characteristics achieved by the optimized method make microbial oil a potential alternative for biodiesel production from yeast at an industrial scale.How to cite: Martinez-Silveira A, Villarreal R, Garmendia G, et al. Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts. Electron J Biotechnol 2018;37. https://doi.org/10.1016/j.ejbt.2018.11.006.     

Stability criteria for certain classes of nonlinear difference equations

The general mth order difference equation X(n+m)+a₁X(n+m?1)+…+a_mX(n) = F[n,X(n),…,X(n+m?1)] is considered. The stability properties of its solutions are studied using the discrete form of Liapunov's direct method. A quadratic form is selected as a possible Liapunov function V(n,X) and a scheme is developed for determining appropriate conditions on this function to insure that its total difference ΔV(n,X) is negative semi-definite or negative definite with respect to the difference equation. The approach is applied to the fourth-order difference equation in full detail to illustrate the method for determining the conditions which imply either uniform stability or uniform asymptotic stability and specific results are obtained. Several comments on, and extensions of, the work done by Puri and Drake for the cases m = 2 and m = 3 are presented.The results of the present approach in the homogeneous case where F[n,X(n),…,X(n+m?1)] = 0 are compared with the usual Schur-Cohn criteria and are shown to be at least as good.     

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid,Myzus persicae

BackgroundPiercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns.ResultsPinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids.ConclusionsThese findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.How to citeUmer N, Naqvi RZ, Rauf I, et al. Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.004.     

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BackgroundThe 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valine-tyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work.ResultsThe alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a β/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C_1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC₅₀ of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively.ConclusionThe inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.How to cite: Espinosa-Hernández E, Morales-Camacho JI, Fernández-Velasco DA, et al. The insertion of bioactive peptides at the C terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity. Electron J Biotechnol 2019;37. https://doi.org/10.1016/j.ejbt.2018.11.001.     

Improved synthesis of the antifungal isobutyl o-coumarate catalyzed by the Aspergillus terreus type B feruloyl esterase

BackgroundHydroxycinnamic acids and some of their derivatives are molecules with interesting biological activities; for instance, hydroxylated hydroxycinnamic esters have proved to have antifungal properties, and thus the generation of these molecules is of industrial importance. In this study, the direct esterification capacity of the pure recombinant type B feruloyl esterase from Aspergillus terreus (AtFAE B) was evaluated by its ability to catalyze the synthesis of isobutyl o-coumarate, an interesting antifungal molecule. A ternary solvent system (isooctane/isobutanol/water) was employed to improve the synthesis of isobutyl o-coumarate, assessing different substrate concentrations, enzyme load, water percentages and pH and temperature values.ResultsAtFAE B showed the highest initial rate at 18% (v/v) isobutanol and 50 mM o-coumaric acid, 0.04 mg/ml of enzyme, 4% (v/v) water without buffer and 40°C. AtFAE B half-lives at 30°C, 40°C and 50°C were 16.5 h, 1.75 h and 3.5 min, respectively. Thus, we decided to evaluate the bioconversion yield at 30°C, where the enzyme showed the highest operational stability. At this temperature, we obtained a yield of ~80% after only 8 h of reaction, using a 78:18:4 isooctane:isobutanol:water ternary solvent system, with 50 mM of o-coumaric acid.ConclusionsUnder these improved conditions, the productivity was 1.06 g isobutyl o-coumarate/L*h with a biocatalyst yield of 209.6 kg isobutyl o-coumarate/kg free AtFAE B, demonstrating the promising potential of AtFAE B to accept the non-canonical o-coumaric acid as the substrate and to achieve the synthesis of isobutyl o-coumarate.How to cite: Vega-Rodríguez AD, Armendáriz-Ruiz MA, Grajales-Hernández DA, et al. Improved synthesis of the antifungal isobutyl o-coumarate catalyzed by the Aspergillus terreus type B feruloyl esterase. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.001     

Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program

BackgroundCultivated peanut (Arachis hypogaea. L) represents one of the most important oil crops in the world. Although much effort has been expended to characterize microsatellites or Simple Sequence Repeats (SSRs) in peanut, the quantity and quality of the markers in breeding applications remain limited. Here, genome-wide SSR characterization and marker development were performed using the recently assembled genome of the cultivar Tifrunner.ResultsIn total, 512,900 microsatellites were identified from 2556.9-Mb genomic sequences. Based on the flanking sequences of the identified microsatellites, 7757 primer pairs (markers) were designed, and further evaluated in the assembled genomic sequences of the tetraploid Arachis cultivars, Tifrunner and Shitouqi, and the diploid ancestral species, A. duranensis and A. ipaensis. In silico PCR analysis showed that the SSR markers had high amplification efficiency and polymorphism in four Arachis genotypes. Notably, nearly 60% of these markers were single-locus SSRs in tetraploid Arachis species, indicating they are more specific in distinguishing the alleles of the A and B sub-genomes of peanut. In addition, two markers closely related with purple testa color and 27 markers near to FAD2 genes were identified, which could be used for breeding varieties with purple testa and high-oleic acid content, respectively. Moreover, the potential application of these SSR markers in tracking introgressions from Arachis wild relatives was discussed.ConclusionsThis study reported the development of genomic SSRs from assembled genomic sequences of the tetraploid Arachis Tifrunner, which will be useful for diversity analysis, genetic mapping and functional genomics studies in peanut.How to cite: Ma J, Zhao Y, Chen H, et al. Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.004.     

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BackgroundProcambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation.ResultsIn this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant.ConclusionsThe high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.How to citeLiu F, Qu Y-K, Geng C, et al. Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.06.007.