Fusion and secretory expression of an exo-inulinase and a d-allulose 3-epimerase to produce d-allulose syrup from inulin.

BACKGROUND: This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION: This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.

Low-Cost Cellulase-Hemicellulase Mixture Secreted by Trichoderma harzianum EM0925 with Complete Saccharification Efficacy of Lignocellulose.

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM0925. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM0925 enzymes have great potential for industrial applications.

GeneHunt for rapid domain-specific annotation of glycoside hydrolases.

The identification of glycoside hydrolases (GHs) for efficient polysaccharide deconstruction is essential for the development of biofuels. Here, we investigate the potential of sequential HMM-profile identification for the rapid and precise identification of the multi-domain architecture of GHs from various datasets. First, as a validation, we successfully reannotated >98% of the biochemically characterized enzymes listed on the CAZy database. Next, we analyzed the 43 million non-redundant sequences from the M5nr data and identified 322,068 unique GHs. Finally, we searched 129 assembled metagenomes retrieved from MG-RAST for environmental GHs and identified 160,790 additional enzymes. Although most identified sequences corresponded to single domain enzymes, many contained several domains, including known accessory domains and some domains never identified in association with GH. Several sequences displayed multiple catalytic domains and few of these potential multi-activity proteins combined potentially synergistic domains. Finally, we produced and confirmed the biochemical activities of a GH5-GH10 cellulase-xylanase and a GH11-CE4 xylanase-esterase. Globally, this "gene to enzyme pipeline" provides a rationale for mining large datasets in order to identify new catalysts combining unique properties for the efficient deconstruction of polysaccharides.

Endoglycosidase S Enables a Highly Simplified Clinical Chemistry Procedure for Direct Assessment of Serum IgG Undergalactosylation in Chronic Inflammatory Disease.

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn's and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry because of a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput. We addressed this issue by using endo-ß-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker toward clinical implementation.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Synergistic action modes of arabinan degradation by exo- and endo-arabinosyl hydrolases.

Two recombinant arabinosyl hydrolases, α-L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endoaction of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the L-arabinose production with high conversion yield.

Possible use of the carbohydrates present in tomato pomace and in byproducts of the supercritical carbon dioxide lycopene extraction process as biomass for bioethanol production.

This study provides information about the carbohydrate present in tomato pomace (skins, seeds, and vascular tissues) as well as in the byproducts of the lycopene supercritical carbon dioxide extraction (SC-CO2) such as tomato serum and exhausted matrix and reports their conversion into bioethanol. The pomace, constituting approximately 4% of the tomato fruit fresh weight, and the SC-CO2-exhausted matrix were enzyme saccharified with 0.1% Driselase leading to sugar yields of ~383 and ~301 mg/g dw, respectively. Aliquots of the hydrolysates and of the serum (80% tomato sauce fw) were fermented by Saccharomyces cerevisiae . The bioethanol produced from each waste was usually >50% of the calculated theoretical amount, with the exception of the exhausted matrix hydolysate, where a sugar concentration >52.8 g/L inhibited the fermentation process. Furthermore, no differences in the chemical solubility of cell wall polysaccharides were evidenced between the SC-CO2-lycopene extracted and unextracted matrices. The deduced glycosyl linkage composition and the calculated amount of cell wall polysaccharides remained similar in both matrices, indicating that the SC-CO2 extraction technology does not affect their structure. Therefore, tomato wastes may well be considered as potential alternatives and low-cost feedstock for bioethanol production.

Assessment of potential enhancing effects of a carbohydrase mixture on phytase efficacy in male broiler chicks fed phosphorus-deficient diets from 1 to 18 days of age.

A total of 540 one-day-old male broiler chicks were used to evaluate the potential enhancing effects of a mixture of 2 commercial carbohydrase preparations on phytase efficacy. A nonphytate phosphorus (nPP) deficient diet (0.15%) was supplemented with different levels of phytase (0X, 1X, 2X, and 3X of recommended level of 500 phytase units per kg of feed), individually or in combination with different levels of a mixture of 2 commercial carbohydrase enzymes [0X, 1X, and 2X of recommended level (X = 0.2 g per kg of feed; a mix with predominantly xylanase and ß-glucanase activity)] to determine their effects on performance and bone development. A standard response curve was developed using graded levels of nPP (0.15 to 0.45%) from dicalcium phosphate in a diet with no phytase supplementation to estimate the phosphorus equivalency of the different enzyme combinations. Each diet was fed to 6 replicate pens of 5 male chicks from 1 to 18 d of age. Phytase at normal usage levels of 500 phytase units per kg of feed was effective (P < 0.05) in improving BW, increasing feed intake, reducing mortality rate, and increasing toe and tibia ash without significantly influencing feed conversion. Addition of extra levels of phytase (2X and 3X) significantly (P < 0.05) improved toe and tibia ash without further affecting performance traits. The carbohydrase mixture failed to improve performance or enhance phytase efficacy. The phosphorus equivalency of phytase was dose dependent, with estimates of 0.100, 0.123, and 0.213% for 1X, 2X, and 3X supplementation levels of phytase, respectively, based on tibia ash. In conclusion, results indicate that increasing the level of phytase resulted in significant improvements in utilization of dietary nPP, whereas the carbohydrase preparation had no significant effects on performance or had limited effects in enhancing phytase ability in young broiler chicks.

Acoustic deposition with NIMS as a high-throughput enzyme activity assay.

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.

Isolation of pectin-enriched products from red beet (Beta vulgaris L. var. conditiva) wastes: composition and functional properties.

The present work was dedicated to the development of an extraction process for red beet (Beta vulgaris L. var. conditiva) by-products that preserves the high molecular weight of the macromolecules with the primary aim of waste upgrading. Our study concerns the extraction of pectin-enriched products with potential thickening properties for their usage in food formulation, as well as with some healthy physiological effect, by using citrate buffer (pH = 5.2) either alone or with enzymes (hemicellulase or cellulase) active on cell wall polysaccharide networks. Considering that red beet tissue contains ferulic acid, which cross-links pectin macromolecules through arabinose residues to anchor them into the cell wall, an alkaline pretreatment was also evaluated in order to perform polysaccharide hydrolysis in the cell wall network to accomplish higher renderings. Chemical composition and yield, as well as the in vitro glucose retention exerted by the isolated fiber products were finally analyzed.

Assessment of commercial hemicellulases for saccharification of alkaline pretreated perennial biomass.

The objective of this research was to measure the effects of different cellulase and hemicellulase mixtures on fermentable sugar production from two different perennial biomasses--switchgrass and a low-impact, high-diversity prairie biomass mixture (LIHD). Each was subjected to NaOH pretreatment, followed by hydrolysis with a commercial cellulase and ß-glucosidase mixture [CB] supplemented with either of two hemicellulases. For both biomasses, there was little gain in sugar yield when using CB alone beyond 20-25 mg/g TS; further gain in yield was possible only through hemicellulase supplementation. An equation that modeled CB and hemicellulase effects as occurring independently fit the data reasonably well, except at the lowest of cellulase loadings with hemicellulase, where synergistic interactions were evident. Examination of the marginal effectiveness of enzyme loadings (incremental grams sugar per incremental mg enzyme) over a broad range of loadings suggests that there is no need to customize enzymatic hydrolysis for NaOH-pretreated switchgrass and LIHD.

Horticultural waste as the substrate for cellulase and hemicellulase production by Trichoderma reesei under solid-state fermentation.

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), beta-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and beta-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.

Isolation and purification of D-mannose from palm kernel.

An economically viable procedure for the isolation and purification of d-mannose from palm kernel was developed in this research. The palm kernel was catalytically hydrolyzed with sulfuric acid at 100 degrees C and then fermented by mannan-degrading enzymes. The solution after fermentation underwent filtration in a silica gel column, desalination by ion-exchange resin, and crystallization in ethanol to produce pure d-mannose in a total yield of 48.4% (based on the weight of the palm kernel). Different enzymes were investigated, and the results indicated that endo-beta-mannanase was the best enzyme to promote the hydrolysis of the oligosaccharides isolated from the palm kernel. The pure d-mannose sample was characterized by FTIR, (1)H NMR, and (13)C NMR spectra.

The glucosinolate-myrosinase system in nasturtium (Tropaeolum majus L.): variability of biochemical parameters and screening for clones feasible for pharmaceutical utilization.

Leaves of Tropaeolum majus L. contain high amounts of the glucosinolate glucotropaeolin. They are used in traditional medicine to treat infections of the urinary tract. When Tropaeolum leaves are consumed, glucotropaeolin is hydrolyzed to yield mustard oils, which are absorbed in the intestine and excreted in the urine, exhibiting their antimicrobial activity. For a corresponding phytopharmacon, a sufficiently high glucotropaeolin concentration is required and any degradation of glucosinolates while drying must be minimized, i.e. the post mortal cleavage by myrosinases, which are activated by ascorbic acid. In extensive screenings, the dominant parameters determining the glucotropaeolin content in the dried leaves were quantified. It turned out that the glucotropaeolin concentration in the dried leaves represented the most suitable screening parameter. The screening of several hundred Tropaeolum plants resulted in the selection of eight high-yield varieties, from which in vitro plants had been generated and propagated as a source for large field trials.

Performing a three-step process for conversion of chitosan to its oligomers using a unique bipolar membrane electrodialysis system.

Chitosan, a linear polysaccharide composed of beta-1,4 linked d-glucosamine residues, can be depolymerized into oligomers by enzymatic reaction with chitosanase. Recently, bipolar membrane electrodialysis (BMED) has been used for chitosan solubilization and for terminating the enzymatic reaction by action of electrogenerated acid and base, respectively. The aim of the present study was to test a complete "3-in-1" process using a three-compartment BMED configuration to perform simultaneously the solubilization of chitosan, the inactivation of chitosanase, and the demineralization of the oligomers. In addition, the BMED process was compared to a conventional process using chemical acid and base. The BMED method was found to be as effective as the conventional method for solubilizing the chitosan and for inactivating the chitosanase. Furthermore, the use of BMED allowed a demineralization rate of 53% of the chito-oligomer solution in the diluate compartment. A global process of chitosan hydrolysis into its oligomers using a BMED system was proposed. This technology has great potential for industrial application in chitosan oligomer preparation, because it is convenient and ecological and it produces chito-oligomers with a lower mineral content compared with the conventional method.

Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding.

To identify the amino acids responsible for the substrate binding of chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), Tyr148 and Lys218 of the chitosanase were mutated to serine and proline, respectively, and the mutated chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.16% of the wild type, respectively. When the (GlcN)3 binding ability to the chitosanase was evaluated by fluorescence spectroscopy and thermal unfolding experiments, the binding abilities of both mutant enzymes were markedly reduced as compared with the wild type enzyme. The affinity of the enzyme for the trisaccharide decreased by 1.0 kcal/mol of binding free energy for Y148S, and 3.7 kcal/mol for K218P. The crystal structure of K218P revealed that Pro218 forms a cis-peptide bond and that the state of the flexible loop containing the 218th residue is considerably affected by the mutation. Thus, we conclude that the flexible loop containing Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less critical, but still important, due to a stacking interaction or hydrogen bond.

Assessment of potential interactions between phytase and glycosidase enzyme supplementation on nutrient digestibility in broilers.

The effects of microbial 3-phytase and glycosidase enzymes, and their interactions, on energy values and nutrient digestibility in diets rich in nonstarch polysaccharides (NSP) were studied in diets based on corn, wheat, or barley. Four diets were prepared with each cereal grain. One had no enzymes, a second had 500 units of phytase, a third had glycosidase enzyme, and a fourth had phytase and glycosidase. The glycosidases used were alpha-galactosidase (corn diet), xylanase (wheat), and beta-glucanase (barley). Glycosidase decreased intestinal viscosity, whereas phytase increased this parameter in corn diets. Phytase increased AME in corn diets, whereas beta-glucanase in barley diets improved AME and AMEn, and digestibility of dry matter, starch, beta-glucans, and lipid. Xylanase in wheat diets improved dry matter and starch digestibility. Phytase increased total phosphorus retention in all diets, and significant interactions between glycosidase enzymes and phytase were detected in wheat and barley diets. Phytase decreased phosphorus excretion in corn and barley diets, whereas alpha-galactosidase increased phosphorus excretion in corn diets. Phytase in corn diets and beta-glucanase in barley diets increased calcium retention, whereas inclusion of xylanase decreased calcium retention in wheat diets. Phytase and beta-glucanase decreased calcium excretion in corn- and barley-based diets, respectively. An interaction was detected between phytase and beta-glucanase in barley diets, in which calcium excretion was reduced. In general, no negative interactions between phytase and glycosidase enzymes were found, indicating that both types of enzymes may be used together in feeds based on corn, wheat, or barley.

In vitro assessment of the enzymatic degradation of several starch based biomaterials.

The susceptibility of starch-based biomaterials to enzymatic degradation by amylolytic enzymes (glucoamylase and alpha-amylase) was investigated by means of incubating the materials with a buffer solution, containing enzymes at different concentrations and combinations, at 37 degrees C for 6 weeks. Two polymeric blends of corn starch with poly(ethylene-vinyl alcohol) copolymer and poly(epsilon-caprolactone), designated by SEVA-C and SPCL, respectively, were studied. The material degradation was characterized by gravimetry measurements, tensile mechanical testing, scanning electron microscopy (SEM), and Fourrier transform infrared-attenuated total reflectance (FTIR-ATR). The degradation liquors were analyzed for determination of reducing sugars, as a result of enzyme activity, and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to identify the degradation products. All of the analysis performed showed that starch polymeric blends are susceptible to enzymatic degradation, as detected by increased weight loss and reducing sugars in solution. alpha-Amylase caused significant changes on the overall mechanical properties of the materials, with a decrease of about 65% and 58% being observed in the moduli for SEVA-C and SPCL, respectively, when compared with the control (samples incubated in buffer only). SEM analysis detected the presence of fractures and pores at the material's surface as a result of starch degradation by amylolytic enzymes. FTIR spectra confirmed a decrease on the band corresponding to glycosidic linkage (-C-O-C-) of starch after incubation of the materials with alpha-amylase. In contrast, the incubation of the polymers in buffer only, did not cause significant changes on the material's properties and morphology. Comparing the two materials, SEVA-C exhibited a higher degradability, which is related to the physicochemical structure of the materials and also to the fact that the starch concentration is higher in SEVA-C. The identification of the degradation products by HPAEC-PAD revealed that glucose was the major product of the enzymatic degradation of starch-based polymers. alpha-Amylase, as expected, is the key enzyme involved in the starch degradation, contributing to major changes on the physicochemical properties of the materials. Nevertheless, it was also found that starch-based polymers can also be degraded by other amylolytic enzymes but in a smaller extent.

Assessment of intestinal permeability: enzymatic determination of urinary mannitol, raffinose, sucrose and lactose on Hitachi analyzer.

The sugar absorption test is the usual test for measurement of intestinal permeability. After intestinal absorption of probe sugars the subsequently excreted sugars are measured in urine. We have developed four enzymatic methods for the measurement of the urinary concentration of the probe sugars mannitol, raffinose, lactose and sucrose. Mannitol, lactose and sucrose are directly measured on Hitachi 917 using mannitol dehydrogenase, beta-galactosidase and invertase, respectively, as enzyme reagents. Raffinose measurement needs a three hours preincubation with alpha-galactosidase, after which the liberated sucrose is measured. The analytical performances such as within- and between-run precision, linearity, lowest detection limit, interference of other sugars and comparison with a gas chromatographic method are described for the four methods. These methods are accurate an can easily be performed in any clinical laboratory.

Enzymes for animal feeding from Penicillium chrysogenum mycelial wastes from penicillin manufacture.

AIMS: Several enzymes from mycelial wastes of Penicillium chrysogenum fungal cultures from penicillin manufacture were studied as supplements for animal feeding. METHODS AND RESULTS: Proteins were precipitated with tannic acid, acetone, ethanol or polyethyleneglycol 6000. The levels of the enzyme activities found in the different precipitates were similar. The activities of invertase, beta-1,3-glucanase, lipase and tannase were higher and those of amylase, cellulase, pectinase, protease and xylanase were lower. Precipitation with tannic acid was selected as an easy and simple method. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: An enzymatic precipitate from these P. chrysogenum wastes, comparable to a commercial enzymatic additive used in animal feeding, was obtained.