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The replacement of expensive components in microbial growth media with pretreated lignocellulosic waste component to increase the product spectrum and add value to the bioproducts has been encouraged to achieve sustainable and feasible utilization of waste biomass as per the biorefinery approach. This study demonstrates an integrated biorefinery approach towards utilization of sugarcane bagasse and biomass of Mucor circinelloides ZSKP. A maximum reducing sugar recovery of 80.67 g/l was achieved after combining pretreatment with saccharification. A low temperature, glycerol, and ammonium phosphate pretreatment method was established, where glycerol pretreatment conditions were reduced from 250 to 150 °C and from 120 to 45 min. The ammonium phosphate-containing hydrolysate yielded 12.89 g/l of fungal biomass after fermentation to add to 20.8 g lignin from the delignification step. The biomass production was further improved to 17.69 g/l after supplementation with corn steep solids and mineral salts. The fermentation process also yielded 2.36 g/l chitosan and 4.9 g/l of lipids after extraction from the oleaginous fungus. The lignin infused glycerol plasticized chitosan biocomposite plastic had a 100% improvement in thermogravimetric properties with almost 50% more energy needed to increase the temperature of the material when compared to glycerol only plasticized biocomposite. The fungal chitosan showed antimicrobial properties and was effective as a preservative spray for fresh tomatoes and apples extending their shelf life to at least 14 and 18 days, respectively. This study therefore demonstrated that a novel two-step pretreatment process could be environmentally beneficial and yielded multiple products for biotechnological applications. [ABSTRACT FROM AUTHOR]

Cowpea cultivation generates large quantities of biomass after pod harvest which are underutilized and could be exploited for energy generation. In this study, evaluation of sugar production by dilute sulfuric acid pretreatment of cowpea haulm for potential ethanolic fermentation was carried out. A central composite design (CCD) was used to investigate the effects of temperature (100–120 °C), time (30–90 min), and acid concentration (1.0–4.0%) on sugar yield and inhibitor formation. The model F-values of 25.42, 78.81, and 6.96 for xylose yield, glucose yield, and total inhibitor concentration, respectively, with low probability value (p < 0.05) suggest a high significance of the models. While the R2-values of 0.9581, 0.9861, and 0.8424 indicated a satisfactory agreement of the quadratic models in predicting the responses. The quadratic model predicted optimum conditions for maximum xylose (76.5%) and glucose yield (28.6%), at minimum inhibitor concentration (2.34 g/L) and temperature (110 °C), with an acid concentration of 3.1% and reaction time of 55 min. These conditions were validated experimentally suggesting the effectiveness of experimental design towards process optimization. The resulting sugar-rich prehydrolysate was detoxified and fermented to ethanol using co-cultures of Saccharomyces cerevisiae BY4743 and Scheffersomyces stipitis (PsY633), while the recovered pretreated solid was subjected to simultaneous saccharification and fermentation with prehydrolysis (PSSF). A total ethanol titer of 15.67 g/L was obtained which corresponds to an overall conversion efficiency of 75%. This suggests that cowpea haulm could also be potentially exploited for bioethanol production either singly or in combination with other lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Five deep eutectic solvents (DES) were screened for their effectiveness in improving biosugar yield in Bambara groundnut haulm (BGH) for bioethanol production. Choline chloride-lactic acid (ChCl-LA) treatment provided the most promising result and was further optimised by investigating the effect of temperature and time on cellulose loss and enzymatic sugar yield. ChCl-LA pretreatment at 100 °C for 1 h was observed to be the best condition for hemicellulose (54.5%) and lignin (60.7%) removal along with optimum sugar recovery of 94.8%. The resulting hydrolysate was concentrated and fermented for 72 h with Saccharomyces cerevisiae BY4743, and a maximum ethanol concentration of 11.57 g/L was achieved with an ethanol yield of 0.38 g/g sugar and productivity of 0.19 g/L/h. From the results obtained, it is evident that deep eutectic solvents are effective for breaking the recalcitrance in BGH due to their high delignification capacity. Furthermore, a recovery of the amorphous glucose and xylose in the BGH prior to DES pretreatment is vital to prevent sugar loss, thereby improving the biosugar yield for bioethanol production. [ABSTRACT FROM AUTHOR]

The effective and efficient valorization of polysaccharide-rich feedstocks is undisputedly a key factor in the quest for a more sustainable planet and a biobased economy. Several covalent and non-covalent interactions within the biomacromolecules define the complex, intricate, and rigid structure of lignocellulose and related biomasses. However, none of the current methods to disrupt the complex interactions of feedstocks and enhance their hydrolysis for subsequent valorization are highly selective and efficient enough, hence the need for new methodologies in this respect. Deep eutectic solvents (DES) are special types of ionic liquids, which are cheaper, less toxic, and more biocompatible than other ionic liquids. They have been identified as important green solvents and functional fluids with immense potential for catalytic transformation of polysaccharide-rich biomass into a diverse range of products. The unique properties of DES enable them to effectively dissolve and convert biomass into valuable end products. This review paper sheds light on the application of DES in the deconstruction and fractionation of different feedstocks, particularly in the pretreatment step. In this regard, it covers the recent advancements in the applications of various DES in the valorization of biomass and its major components (i.e., cellulose, xylan, chitin, starch, pectin) with special emphasis on the relationship between the inherent properties of DES and their dissolution potential. Focus is also placed on the toxicity, biocompatibility, and reusability of DES. Finally, this article addresses the current and potential issues in the industrial application of DES, with a special focus on future opportunities. [ABSTRACT FROM AUTHOR]

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved. [ABSTRACT FROM AUTHOR]

A new aminopeptidase (An-APa) was identified and biochemically characterized from Aspergillus niger CICIM F0215. It had maximal activity at 40 °C and pH 7.0 and exhibited a broad substrate specificity both on hydrophilic and hydrophobic amino acid residues at N-terminals. With An-APa hydrolysis for 1 h, the casein-pepsin and soybean protein isolates (SPI)-pepsin hydrolysates released both hydrophilic and hydrophobic amino acids and the hydrophobic amino acids having Q values (degree of hydrophobicity) greater than 1500 cal/mol were remarkably released. Leu, Ile, Phe, Tyr, Trp, Pro, Val and Lys in the casein hydrolysate after treatment with An-APa increased 18.61, 0.84, 11.35, 13.18, 3.34, 6.30, 7.46, and 8.19 mg/100 mL, respectively, and 19.72, 1.47, 18.37, 11.72, 4.61, 4.10, 8.13, and 5.85 mg/100 mL, respectively, in the SPI hydrolysate. Both accounted for 65.0% and 64.4% of total released free amino acids from casein and SPI hydrolysates, respectively. This indicated that An-APa could be potentially applicable in debittering protein hydrolysates. [ABSTRACT FROM AUTHOR]

Two new aspartic proteases, PepAb and PepAc (encoded by pepAb and pepAc), were heterologously expressed and biochemically characterized from Aspergillus niger F0215. They possessed a typical structure of pepsin-type aspartic protease with the conserved active residues D (84, 115), Y (131, 168) and D (281, 326), while their identity in amino acid sequences was only 19.0%. PepAb had maximum activity at pH 2.5 and 50 °C and PepAc at 3.0 and 50 °C. The specific activities of PepAb and PepAc toward casein were 1368.1 and 2081.4 U/mg, respectively. Their activities were significantly promoted by Cu2+ and Mn2+ and completely inhibited by pepstatin. PepAb exhibited higher catalytic efficiency (kcat/Km) toward soy protein isolates than casein, while PepAc showed higher catalytic efficiency toward casein. The hydrolysis capacities of PepAb and PepAc on soy protein isolates were slightly lower than that of previously identified A. niger aspartic protease, PepA (aspergillopepsin I), while the resultant peptide profiles were remarkably different for all three proteases. [ABSTRACT FROM AUTHOR]

Objectives: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery.Results: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate.Conclusions: By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate. [ABSTRACT FROM AUTHOR]

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions. [ABSTRACT FROM AUTHOR]

α-Glucosidase was immobilized onto an epoxy-activated resin (Eupergit C) to catalyze maltose into isomaltooligosaccharides (IMO), and then the effects of organic-aqueous media on the enzymatic properties of immobilized α-glucosidase were examined. An immobilization efficiency of 79.61% was obtained under the condition of pH 6.0, ionic strength of 2.0 M, and 30 mg of protein/g of resin. The butyl acetate-aqueous biphasic system was found to significantly improve the catalytic activity of the immobilized enzyme and the yield of IMO. The highest yield of IMO (50.83%, w/w) was obtained at pH 4.5 and 55 °C in a butyl acetate/buffer system (25:75, v/v). In addition, the immobilized enzyme particles were distributed into the organic phase after the completion of transglycosylation, which facilitates the separation and recycling use of the immobilized enzyme. Immobilized α-glucosidase retains a robust reusability in this continuous operation model. The present findings are of potential in improving the IMO manufacturing process. [ABSTRACT FROM AUTHOR]

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40-80 °C) and pH (2.0-6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K , V , K , and K / K of the pure phytase were 0.21 mM, 131.58 nmol mg s, 1.64 × 10 s, and 7.81 × 10 M s, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca, Mg and Mn, but inhibited by Zn, Cu, Fe, Pb, Ba and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry. [ABSTRACT FROM AUTHOR]

The biocontrol of Listeria monocytogenes on fresh-cut tomato using nisin and organic acids was investigated. Fresh-cut samples inoculated with 10 CFU/mL of L. monocytogenes, treated with nisin (5,000 IU/mL), a combination of nisin and organic acids (acetic and citric acids at 3 and 5% each), with chlorine at 200 ppm as a control, and stored for six days at 4, 10, and 25°C were used to evaluate certain physicochemical qualities (pH, titratable acidity, soluble solid content, vitamin C content, and color). Nisin treatment significantly ( p<0.05) reduced bacterial population by 1.91-3.07 log CFU/mL. Nisin-citric acid combination provided 2.65-3.29 log CFU/mL reduction, while nisin-acetic acid combination provided 2.93-4.15 log CFU/mL reduction. The control treatment provided <1-2 fold log reductions. Slight variations in physicochemical properties of fresh-cut tomato were observed. Nisin and organic acids can be used to improve the microbial safety of fresh-cut tomato. [ABSTRACT FROM AUTHOR]

Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application. [ABSTRACT FROM AUTHOR]

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 ( ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli. [ABSTRACT FROM AUTHOR]

In order to rationally manipulate the cellular metabolism of Escherichia coli for d-lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase ( ackA), phosphotransacetylase ( pta), phosphoenolpyruvate synthase ( pps), pyruvate formate lyase ( pflB), FAD-binding d-lactate dehydrogenase ( dld), pyruvate oxidase ( poxB), alcohol dehydrogenase ( adhE), and fumarate reductase ( frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA- pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 ( ackA- pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 ( ackA- pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l d-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of d-lactate production are among the highest reported and the results have revealed which genetic manipulations improved d-lactate production by E. coli. [ABSTRACT FROM AUTHOR]

The combined effect of simultaneous saccharification and fermentation and separate hydrolysis and fermentation (SHF) for ethanol production by Kluyveromyces marxianus 6556 was studied using two lignocellulosic feedstocks viz., corncob and soybean cake. The ethanologenic efficiency of K. marxianus 6556 was observed as 28% (theoretical yield) in a fermentation medium containing glucose, but, there was no ethanol production by cells grown on xylose. A maximum sugar release of 888 mg/g corncob and 552 mg/g soybean cake was achieved through acid hydrolysis pretreatment. Furthermore, corncob and soybean cake treated with commercial cellulase (100 IU for 48 h) from Trichoderma reesei yielded reducing sugars of 205 and 100 mg/g, respectively. Simultaneous saccharification and fermentation resulted in highest ethanol production of 5.68 g/l on corncob and 2.14 g/l on soybean cake after 48 h of incubation. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the hydrolysates obtained through SHF of corncob and soybean cake. [ABSTRACT FROM AUTHOR]

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t-1 pulp. [ABSTRACT FROM AUTHOR]

To find a lipase for synthesis of flavor esters in food processing, a total of 35 putative lipases from Aspergillus niger F0215 were heterologously expressed and their esterification properties in crude preparations were examined. One of them, named An-lipase with the highest esterification rate (23.1%) was selected for further study. The purified An-lipase had the maximal activity at 20 °C and pH 6.5 and the specific activity of 1293 U/mg. Sixty percent of the activity was maintained in a range of temperatures of 0–30 °C and pHs of 3.0–8.5. The highest hydrolysis activity of An-lipase was towards pNPC (C8), followed by pNPB (C4) and pNPA (C2), then pNPL (C12). Km, Vmax, kcat, and kcat/Km towards pNPC were 26.7 mmol/L, 129.9 mmol/(L h), 23.2 s−1, and 0.8/mM/s, respectively. The ethyl lactate, butyl butyrate, and ethyl caprylate flavor esters were produced by esterification of the corresponding acids with conversion efficiencies of 15.8, 37.5, and 24.7%, respectively, in a soybean-oil-based solvent system. In conclusion, An lipase identified in this study significantly mediated synthesis of predominant flavor esters (ethyl lactate, butyl butyrate, and ethyl caprylate) in a soybean-oil-lacking other toxic organic solvents, which has potential application in food industries. [ABSTRACT FROM AUTHOR]