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Japanese quail (Coturnix coturnix japonica) is a popular experimental animal model in scientific research. The present study investigated the effects of dietary multiple enzyme supplementation on growth performance, carcass characteristics, nutrient digestibility and small intestinal histomorphology in quails fed diets based on wheat and soya bean meal. A total number of 192 1-day-old quails were assigned to three treatments with 16 replicates in each and four quails per replicate for 38 days. The control group received a basal diet, and the treatment groups received a basal diet with 0.10 or 0.20% multi-enzyme, respectively. Growth performance parameters, carcass characteristics, nutrient digestibility and small intestinal histomorphology in quails were evaluated. Dietary supplementation of multi-enzymes to diet significantly increased body weight gain and improved the feed conversion rate. Moreover, quails fed with 0.10 or 0.20% multi-enzymes showed better ash digestibility coefficients and apparent metabolizable energy coefficients than the control quails. Furthermore, quails fed on a diet containing 0.20% multi-enzyme had the highest crude fiber digestibility. The villi length and the villi length/crypt depth ratio of the duodenum were significantly increased and the crypt depth was decreased in quails-fed diets supplemented with both multi-enzyme levels. However, feed consumption, carcass yield, carcass weight, heart weight, gizzard weight, liver weight and total intestine weights were not affected by treatments. In conclusion, our results showed that dietary supplementation of multi-enzymes to a wheat and soybean meal-based diet enhanced the growth performance and nutrient digestibility of quails. [ABSTRACT FROM AUTHOR]

Artificial multi-enzyme cascades bear great potential for bioconversion of C1 compounds to value-added chemicals. Over the past decade, massive efforts have been devoted to constructing multi-enzyme cascades to produce glycolic acid, rare functional sugars and even starch from C1 compounds. However, in contrast to traditional fermentation utilizing C1 compounds with the expectation of competitive economic performance in future industrialization, multi-enzyme cascades systems in the proof-of-concept phase are facing the challenges of upscaling. Here, we offered an overview of the recent advances in the construction of in vitro multienzyme cascades and whole-cell transformation using C1 compounds as substrate. In addition, the existing challenges and possible solutions were also discussed aiming to combine the strengths of in vitro and in vivo multi-enzyme cascades systems for upscaling. [ABSTRACT FROM AUTHOR]

This work has been focused on developing a novel biosensor assisted by multivariate calibration methods to determine triglycerides (TGs, triacetin, tributyrin, tricaproin, tricaprylin, and tricaprin) in lyophilized serum samples. To achieve this goal, a bare glassy carbon electrode (GCE) was modified and used as the biosensing platform. To increase the sensitivity of the developed method, hydrodynamic methods were used to calibrate the biosensor response. To increase the selectivity of the developed biosensor, it was assisted by partial least squares-1 (PLS-1), radial basis function-PLS (RBF-PLS), and RBF-artificial neural network (RBF-ANN) for exploiting firstorder advantage. After characterization of the modifications, the first-order advantage was exploited to increase the selectivity of the method by building a multivariate calibration set in a pre-analyzed lyophilized serum with different TGs concentrations, which were chosen according to the individual calibration curve. Calibration models were then built in the same pre-analyzed lyophilized serum and analyzed by PLS-1, RBF-PLS, and RBFANN. Therefore, their performances were examined to predict concentrations of a validation set. The results confirmed the successfulness of the calibration model developed by RBF-ANN. Finally, it was used to analyze two serum samples, and the results demonstrated that the method was successful because its results were compared with a reference method. [ABSTRACT FROM AUTHOR]

Enzymes are outstanding (bio)catalysts, not solely on account of their ability to increase reaction rates by up to several orders of magnitude but also for the high degree of substrate specificity, regiospecificity and stereospecificity. The use and development of enzymes as robust biocatalysts is one of the main challenges in biotechnology. However, despite the high specificities and turnover of enzymes, there are also drawbacks. At the industrial level, these drawbacks are typically overcome by resorting to immobilized enzymes to enhance stability. Immobilization of biocatalysts allows their reuse, increases stability, facilitates process control, eases product recovery, and enhances product yield and quality. This is especially important for expensive enzymes, for those obtained in low fermentation yield and with relatively low activity. This review provides an integrated perspective on (multi)enzyme immobilization that abridges a critical evaluation of immobilization methods and carriers, biocatalyst metrics, impact of key carrier features on biocatalyst performance, trends towards miniaturization and detailed illustrative examples that are representative of biocatalytic applications promoting sustainability. [ABSTRACT FROM AUTHOR]

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the Acore domains, yet domain interfaces and the flexible Asub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of multienzyme+probiotic (MEP) supplementation on carcass characteristics and serum biochemistry of broiler chickens fed fermented shea butter cake meal (FSBCM) based diets. A total of two hundred and sixty (260), 28-day-old broiler chicks were distributed into four treatments, with five replicates of 13 birds each using a completely randomized design and reared on deep litter system from 28 to 56 days of age. The four treatments were: positive control - T1 (corn-soybean based diet); negative control - T2 (20% FSBCM replacement for corn in T1); diet 3 - T3 (T2 diet + 0.05% MEP) and diet 4 - T4 (T2 diet + 0.10% MEP). On day 56, two birds per each pen were selected for carcass and organ evaluation and other two birds per replicate were selected for serum biochemical analysis. The results indicated that addition of MEP to FSBCM diet caused a significant (p<0.05) increase in live weight, dressing percentage and breast meat yield than FSBCM diet without MEP and did not differ from the T1 diet. The abdominal fat in birds fed MEP treated diets were significantly (p<0.05) lower than those groups fed PC and FSBCM diets. Serum glucose was similar (p>0.05) between birds fed PC and MEP treated diets but significantly (p<0.05) higher than those on FSBCM diet, whereas serum cholesterol, alanine aminotransferase and aspartate aminotransferase concentrations reduced significantly (p<0.05) in groups fed MEP supplemented FSBCM diets. All other parameters were not significantly (p>0.05) affected by the MEP supplementation. Therefore, MEP supplementation in FSBCM based diet improved live weights and carcass attributes without any detrimental effect on blood composition of the broiler chickens. [ABSTRACT FROM AUTHOR]

This study was executed to investigate the effect of supplementing three multienzyme levels (0, 0. 1, and 0.2%) with two types of diet [standard diet (SD) vs. low-density diet (LDD)] on immune response, blood hematology and biochemistry, antioxidant status, and organ histology of broilers during 1–38 days of age. A total of 216 unsexed 1-day-old Arbor Acres broiler chicks were randomly distributed, on a factorial design (2 × 3), to six treatments each with six replicates. There were six chicks per replicate. Results showed that LDD significantly decreased body weight gain (BWG) of broilers, but did not affect the European Production Efficiency Index (EPEI). Addition of multienzymes at both levels (0.1 and 0.2%) significantly increased BWG and improved EPEI, compared to the control diet. Alanine aminotransferase (ALT), aspirate aminotransferase (AST), malondialdehyde (MDA), lymphocyte, lymphocyte transformation test (LTT), and phagocyte activity (PA) were significantly higher for LDD than the SD, but eosinophil was lower. Supplementation of multienzymes significantly decreased ALT, AST, and MDA, compared to the control group, but increased packed cell volume (PCV), hemoglobin (Hgb), lymphocytes, and monocytes. Immune organs, such as spleen, thymus, and the bursa of Fabricius were significantly increased with multienzyme supplementation. It could be concluded that multienzyme supplementation at either 0.1 or 0.2% to SD or LDD improved EPEI and immune status of broiler chicks.

[Display omitted] An enantiopure (2 R ,5 R)-1,3-oxathiolane was obtained using a multienzymatic cascade protocol. By employing a combination of surfactant-treated subtilisin Carlsberg and Candida antarctica lipase B, the absolute configuration of the resulting 1,3-oxathiolane ring was efficiently controlled, resulting in an excellent enantiomeric excess (>99%). This enantiopure 1,3-oxathiolane derivative is a key precursor to anti-HIV agents, such as lamivudine, through subsequent N -glycosylation. [ABSTRACT FROM AUTHOR]

Graphical abstract We presented a simple and efficient strategy based on DNA scaffold for encapsulation multienzyme into ZIF-8, which can efficiently suppress enzyme leaching and significantly improve the enzymatic activity and stability. Highlights • The DNA cross-linking enzyme was prepared and encapsulated into ZIF-8. • The DNA scaffold could suppress enzyme leaching and shorten distance of bienzyme. • The prepared bienzyme enhanced catalytic efficiency, stability, and reusability. • The prepared bienzyme could detect low level of glucose with better selectivity. Abstract The encapsulated enzyme system by metal–organic frameworks exhibited great potential in pharmaceuticals, enzyme fuel cells, and biocatalysis. However, the enzymatic activity and overall catalytic efficiency are severely hampered due to their enzyme leaching and causal enzyme location. Herein, for the first time, horseradish peroxidase (HRP) and glucose oxidase (GOx) used as model enzymes were cross-linked by rationally designed DNA scaffold network, and the multienzyme system has been successfully encapsulated into zeolite imidazolate framework-8 (ZIF-8). The prepared encapsulation multienzyme can efficiently suppress enzyme leaching from the MOFs skeleton, and the leaching amount was more than 10-times lower than the traditional MOFs encapsulation strategy. The well-tailored proximity and colocalization of cascade enzymes by the DNA scaffold network obviously improved the overall catalytic efficiency, enzymatic activity, and kinetic performance. The K m and k cat / K m for encapsulation multienzyme was 13.8 mM and 3.47 s−1 mM−1, which was 0.27-fold lower and 9.9-fold higher than those of free enzymes, respectively, indicating a better substrate affinity. Furthermore, the multienzyme system exhibited excellent stability and reusability compared with free enzymes and control groups, which could preserve 95% initial activity at 50 °C after 75 min incubation and more than 87% of the original activity after 14 cycles. Finally, the immobilized multienzyme exhibited enhanced performance to detect low concentration of glucose (0.4 µM) with excellent selectivity. The outcome indicated that the encapsulated multienzyme could be extended for broad practical applications in biotechnology, biocatalysis, and biomedical engineering. [ABSTRACT FROM AUTHOR]

The present study was conducted to test whether a multi-enzyme produced by a fungus would keep performance of broiler chicks receiving a diet with almost 3% less nutrients (energy, protein, amino acids, calcium and phosphor) under two different housing density. In both experiments, positive control received standard broiler diet, negative control received a diet with 3% less nutrients than standard broiler diet and enzyme group received the negative control diet+supplemental enzyme. In the first trial with 1000, day-old chicks housed 10 birds/m2, only 1% improvement in feed conversion efficiency and body growth was achieved with enzyme supplementation in contrast to the negative control at the end of 35 days feeding period. In the second trial with 1200, day-old-chicks accommodated 16 birds/m2. The results showed that enzyme improved broiler performance almost 3.5% in contrast to the performance obtained from the negative control. In conclusion, supplementing broiler diets based on maize/soya with a multi-enzyme produced by a single fungus provides a great potential to gain the gap created by formulating almost 3% nutrients reduction in broilers housed with 16 birds/m2 density. However, providing high comfort and better rearing condition as the birds at 10 birds/m2 density, positive effects of the enzyme was limited. [ABSTRACT FROM AUTHOR]

The pyruvate dehydrogenase complex (PDC) is a multienzyme assembly that converts pyruvate to acetyl-CoA. As pyruvate and acetyl-CoA play central roles in cellular metabolism, understanding PDC regulation is pivotal to understanding the larger metabolic network. The activity of mammalian PDC is regulated through reversible phosphorylation governed by at least four isozymes of pyruvate dehydrogenase kinase (PDK). Deciphering which kinase regulates PDC in organisms at specific times or places has been challenging. In this study, we analyzed mouse strains carrying targeted mutations of individual isozymes to explore their role in regulating PDC activity. Analysis of protein content of PDK isozymes in major metabolic tissues revealed that PDK1 and PDK2 were ubiquitously expressed, whereas PDK3 and PDK4 displayed a rather limited tissue distribution. Measurement of kinase activity showed that PDK1 is the principal isozyme regulating hepatic PDC. PDK2 was largely responsible for inactivation of PDC in tissues of muscle origin and brown adipose tissue (BAT). PDK3 was the principal kinase regulating pyruvate dehydrogenase activity in kidney and brain. In a well-fed state, the tissue levels of PDK4 protein were fairly low. In most tissues tested, PDK4 ablation had little effect on the overall rates of inactivation of PDC in kinase reaction. Taken together, these data strongly suggest that the activity of PDC is regulated by different isozymes in different tissues. Furthermore, it appears that the overall flux through PDC in a given tissue largely reflects the properties of the PDK isozyme that is principally responsible for the regulation of PDC activity in that tissue. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Multienzymes were assembled to cascade system for alkene production. • Multienzymes were assembled with controllable ratio and position of each enzyme. • Multienzymes exhibited obvious substrate channeling. • Multienzymes showed notably improved tolerance and reusability. Abstract We develop an efficient and economic cascade multienzymes for fatty alkene bioproduction based on the lipase hydrolysis coupled to the P450 decarboxylation in the form of multiple enzyme complex. One step preparation of a multienzyme complex was based on a mixture of cell extracts including dockerin-enzyme fusions and one cohesin-cellulose binding module (CBM) fusion through high specific interaction of dockerin and cohesin. Simultaneously, the CBM was bound to cellulose carrier to form co-immobilized multienzyme. The key factors affecting overall efficiency of alkene bioproduction including substrate channeling of hydrolysis and decarboxylation, the ratio and position of two enzymes, stability were all addressed by genetically engineering of the synthetic CBM-cohesin fusions. The multienzymes exhibited more than 9.2 fold enhancement in initial reaction rate and much higher conversion yields (69%–72%) compared to mixture of free enzyme counterpart. The enzymatic cascade based multienzymes could efficiently convert renewable triglycerides to alkenes. [ABSTRACT FROM AUTHOR]

The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o-hE2o, hE1o-hE3, and hE2o-hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them. We carried out multiple physico-chemical studies, including fluorescence, hydrogen-deuterium exchange MS (HDX-MS), and chemical cross-linkingMS(CL-MS). Our fluorescence studies suggested a strong interaction for the hE1o-hE2o subcomplex, but a much weaker interaction in the hE1o-hE3 subcomplex, and failed to identify any interaction in the hE2o-hE3 subcomplex. The HDX-MS studies gave evidence for interactions in the hE1o- hE2o and hE1o-hE3 subcomplexes comprising full-length components, identifying: (i) the N-terminal region of hE1o, in particular the two peptides 18YVEEM22 and 27ENPKSVHKSWDIF 39 as constituting the binding region responsible for the assembly of the hE1o with both the hE2o and hE3 components into hOGDHc, an hE1 region absent in available X-ray structures; and (ii) a novel hE2o region comprising residues from both a linker region and from the catalytic domain as being a critical region interacting with hE1o. The CL-MS identified the loci in the hE1o and hE2o components interacting with each other. [ABSTRACT FROM AUTHOR]

The main objectives of this study were to elucidate the effect of multienzyme and/or probiotics supplementation on performance, antioxidant status, some blood biochemical parameters and immune responses of broiler chickens. A total of 120, unsexed day-old, broiler chicks were randomly assigned into four groups of 30 chicks in five replicates, six birds each. The first group was used as control and fed the basal diet while, chicks in the 2nd,3rd and 4th groups were fed the basal diet supplemented with multi enzymes (Avizyme at 0.20 g/kg diet), probiotics (Biacton at 0.50 g/kg diet) either singly or in combination. Results showed that live body weight and body weight gain were significantly improved by dietary treatments compared to control group. All treatments had significantly increased dressing and total edible parts (%) and decreased abdominal fat, with the best results being achieved by the combined multi-enzyme and probiotic supplementation. Blood constituents (plasma proteins and lipid fractions, thyroid hormones, especially T3 and glucose) were significantly affected by treatments. There are significant improvements in hematological traits including RBCs and WBCs counts, hemoglobin concentration, PCV (%), MCV, MCH and lymphocytes (%). Antioxidant status indices and enzymes including TAC, GSH, and SOD did not significantly affected by different treatments, however, glutathione peroxidase (GPX) activity was significantly higher in broilers fed the supplemented diets compared to the control one. Immune responses in terms of the main lymphoid organs weight (Bursa and Thymus), immunoglobulins (α–globulin, β–globulin, γ- globulin, IgA, and IgG), Phagocytic activity (PA), Phagocytic index (PI), lysozyme activity (LA), Bactericidal activity (BA) and Lymphocyte transformation test (LTT) were significantly improved by treatments compared with the control. It is concluded that a mixture of 0.2g of multi-enzyme plus 0.5g of probiotics /kg diet could be used to improve growth traits and enhance immunity of broiler chicks. [ABSTRACT FROM AUTHOR]

Covering: up to mid of 2018 Type I fatty acid synthases (FASs) are giant multienzymes catalyzing all steps of the biosynthesis of fatty acids from acetyl- and malonyl-CoA by iterative precursor extension. Two strikingly different architectures of FAS evolved in yeast (as well as in other fungi and some bacteria) and metazoans. Yeast-type FAS (yFAS) assembles into a barrel-shaped structure of more than 2 MDa molecular weight. Catalytic domains of yFAS are embedded in an extensive scaffolding matrix and arranged around two enclosed reaction chambers. Metazoan FAS (mFAS) is a 540 kDa X-shaped dimer, with lateral reaction clefts, minimal scaffolding and pronounced conformational variability. All naturally occurring yFAS are strictly specialized for the production of saturated fatty acids. The yFAS architecture is not used for the biosynthesis of any other secondary metabolite. On the contrary, mFAS is related at the domain organization level to major classes of polyketide synthases (PKSs). PKSs produce a variety of complex and potent secondary metabolites; they either act iteratively (iPKS), or are linked via directed substrate transfer into modular assembly lines (modPKSs). Here, we review the architectures of yFAS, mFAS, and iPKSs. We rationalize the evolution of the yFAS assembly, and provide examples for re-engineering of yFAS. Recent studies have provided novel insights into the organization of iPKS. A hybrid crystallographic model of a mycocerosic acid synthase-like Pks5 yielded a comprehensive visualization of the organization and dynamics of fully-reducing iPKS. Deconstruction experiments, structural and functional studies of specialized enzymatic domains, such as the product template (PT) and the starter-unit acyltransferase (SAT) domain have revealed functional principles of non-reducing iterative PKS (NR-PKSs). Most recently, a six-domain loading region of an NR-PKS has been visualized at high-resolution together with cryo-EM studies of a trapped loading intermediate. Altogether, these data reveal the related, yet divergent architectures of mFAS, iPKS and also modPKSs. The new insights highlight extensive dynamics, and conformational coupling as key features of mFAS and iPKS and are an important step towards collection of a comprehensive series of snapshots of PKS action. [ABSTRACT FROM AUTHOR]

Abstract: The elevated value of α‐keto acids has pushed scientists to explore more efficient and less expensive alternatives for their synthesis. In this work, an immobilized tri‐enzyme system that produced α‐keto acids in “one‐pot” from l‐ or racemic mixtures of diverse amino acids was presented. The system combined a broad‐spectrum amino acid racemase (BsrV), a d‐amino acid oxidase (DAAO) and catalase (CAT). BsrV racemized l‐amino acids into their d‐enantiomers, DAAO catalyzed the stereospecific oxidative deamination of the d‐amino acids into their corresponding α‐keto acids, ammonium ion, and H2O2. Finally, CAT converted the inactivating H2O2 into H2O and O2, which can be reused by the oxidase reaction. BsrV thermal stability was improved 3,300‐fold by immobilizing the enzyme on glyoxyl‐activated agarose beads. DAAO and CAT were co‐immobilized on agarose beads functionalized with glutaraldehyde groups for enhancing their stabilities and eliminating H2O2 in a much more effective way. To show the versatility of this system, racemic mixtures of amino acids were converted in their corresponding α‐keto acids. The coupling of the three immobilized enzymes permitted conversions of approximately 99 % through a dynamic kinetic resolution process. This system conserved 100 % of its initial effectiveness after 8 reaction cycles. Collectively, our innovative tri‐enzyme system for the synthesis of α‐keto acids opens the door for a cheapening in the production of many pharmaceutical and cosmetics. [ABSTRACT FROM AUTHOR]

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated. [ABSTRACT FROM AUTHOR]

Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability. Multi-enzyme catalysis, which can accomplish the tandem synthesis of valuable materials/chemicals from renewable feedstocks, establishes a bridge between single-enzyme catalysis and whole-cell catalysis. Multi-enzyme catalysis occupies a unique and indispensable position in the realm of biological reactions for energy and environmental applications. Two complementary strategies, i.e., compartmentalization and substrate channeling, have been evolved by living organisms for implementing the complex in vivo multi-enzyme reactions (MERs), which have been applied to construct multi-enzyme catalytic systems (MECSs) with superior catalytic activity and stabilities in practical biocatalysis. This tutorial review aims to present the recent advances and future prospects in this burgeoning research area, stressing the features and applications of the two strategies for constructing MECSs and implementing in vitro MERs. The concluding remarks are presented with a perspective on the construction of MECSs through rational combination of compartmentalization and substrate channeling. [ABSTRACT FROM AUTHOR]

Biocatalytic C–H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an “Artificially Bioamination Pathway” for C–H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450 BM3 mutant, an alcohol dehydrogenase Sc CR from Streptomyces coelicolor and an amine dehydrogenase Es LeuDH from Exiguobacterium sibiricum , constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes. [ABSTRACT FROM AUTHOR]

Cold-pressed camelina cake (CPCC) is a fibrous co-product of camelina seed pressing and available for livestock feeding. However, information is lacking on the effect of supplementing fiber-degrading enzymes to CPCC-based diets on nutrient utilization by pigs. Experiment 1 determined the effect of multienzyme supplementation on standardized ileal digestibility (SID) of amino acid (AA) and net energy (NE) value of CPCC for pigs. Six ileal-cannulated barrows (average initial body weight [BW] = 36 kg) were fed five diets in 5 x 5 Latin square design with 1 added column to give six replicates per diet. The diets were a corn-soybean meal (SBM)-soybean oil-based diet and the basal diet with corn, SBM, and soybean oil replaced by 25% CPCC with or without multienzyme (600 U of xylanase, 75 U of glucanase, 250 U of cellulose, 30 U of mannanase, 350 U of invertase, 2,500 U of protease, and 6,000 U of amylase/ kg of diet; Superzyme-CS, 0.5 g/kg) in a 2 x 2 factorial arrangement. The fifth diet was a low-casein cornstarch-based diet. The ratio of corn to SBM and soybean oil in the basal diet was identical to the CCPC-containing diets to allow calculation of nutrient digestibility of CPCC by the difference method. On a dry matter (DM) basis, CPCC contained 42% crude protein, 10.5% ether extract, 25.4% neutral detergent fiber (NDF), 2.07% Lys, 0.73% Met, 1.64% Thr, 0.51% Trp, and 22.1 trypsin inhibitor units per milligram, respectively. The SID of Lys, Met, Thr, and Trp for CPCC were 43.5%, 70.7%, 44.8%, and 55.3%, respectively. The digestible energy (DE) and NE values for CPCC were 3,663 and 2,209 kcal/kg of DM, respectively. Multienzyme supplementation did not affect the SID of AA, and DE and NE values for the corn-SBM-CPCC-based diet, and for the CPCC. In experiment 2, the effects of multienzyme dosage (0.5 or 50 g/kg of treated feedstuff) on porcine in vitro digestibility of DM (IVDDM) of CPCC was determined. The IVDDM of CPCC was increased (P < 0.001) with an increase in multienzyme dosage. Multienzyme at 0.5 g/kg did not affect IVDDM of CPCC. However, multienzyme at 50 g/kg increased (P < 0.01) IVDDM for CPCC by at least 16%. In conclusion, multienzyme at 0.5 g/kg did not affect SID of AA and NE values, and IVDDM for CPCC. However, multienzyme at 50 g/kg improved IVDDM of CPCC, implying that the efficacy of the multienzyme with regard to improving nutrient digestibility of CPCC in pigs is dosage dependent. [ABSTRACT FROM AUTHOR]

The multienzyme co-immobilization systems with high cascade catalytic efficiency and selectivity have attracted considerable attention. In this study, through DNA-directed immobilization (DDI) technology, two model enzymes, glucose oxidase (GOD) and horseradish peroxide (HRP) were co-immobilized on the multifunctional silica nanoparticles (DDI enzyme). In addition to the directional distribution promoted by DNA complementary chains, the multienzyme system allowed the control of the stoichiometric ratio of the enzymes by adjusting the ratio of amino/carboxyl groups. The optimal mole ratio of GOD/HRP was 1:2, while the protein loading amount could reach 8.06 mg·g−1. Compared with the conventional direct adsorption, the catalytic activity of the DDI enzyme was 2.49 times higher. Moreover, with the enhancement of thermal and mechanical stability, the DDI enzyme could still retain at least 50% of its initial activity after 12 cycles. Accompanied by an excellent response and good selectivity, the constructed multienzyme systems simultaneously showed the potential as a glucose detector. Therefore, based on the DDI technology, the highly efficient multienzyme co-immobilization system could be further extended for a wider range of research fields. [Display omitted] • Succinic anhydride-chitosan functionalized SiO 2 nanoparticles were prepared. • The protein loading amount of DDI enzyme could reach 8.06 mg·g−1. • The DDI enzyme could retain more than 50% of the initial activity after 12 cycles. • The DDI enzyme possessed good thermostability, reusability and catalytic activity. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the continuous or intermittent impact of a multienzyme supplement on growth performance, nutrient digestibility, and blood metabolites of broilers, and to evaluate production index of dietary supplementation. A total of 315 unsexed day-old Arbor Acres broiler chicks were randomly distributed to seven treatments groups, keeping initial body weights similar, in 35-floor pens (replicates) of nine chicks per replicate (pen) and five experimental units per treatment. All experimental groups were fed the same basal diet and administered seven multienzyme treatments: the 1st group (control) did not receive any enzyme supplementation; the 2nd, 3rd, and 4th groups were administered multienzymes at 0.5, 1.0, and 1.5 mL/L drinking water, respectively. Each enzyme supplemented-group was divided into two subgroups, with additives being applied either continuously (24 h/day) or intermittently (12 h/day) from 1 to 35 days old. Regardless of administration method, multienzyme supplements at 1.0 mL/L water along with a corn-soybean meal diet increased the body weight gain (BWG) by 7.8% compared to 0.5 mL/L water during days 1−21 of age. In addition, 1.5 mL/L water significantly improved BWG by 5.1% of broilers compared to 0.5 mL/L water during days 1−35 of age. Enzyme supplementation at 1.5 mL/L water significantly enhanced feed conversion ratio (FCR) by 4.3% during days 1−21 of age, and FCR by 5.2% and European production index (EPI) by 10.4% during days 1−35 of age compared to the group on 0.5 mL/L water. For the whole period, there were improvements of beneficial consideration in BWG (4.0%), FCR (4.0%), and European production index (8.2%) due to continuous multienzyme supplementation at 0.5 mL/L water compared to the same dose added intermittently. A similar trend was observed due to intermittent multienzymes at 1 mL/L drinking water that resulted in increased BWG by (6.4%) and improved FCR by (6.7%) and EPI by (12.7%). Intermittent administration significantly increased feed intake of broilers during 22−35 days of age compared to continuous supplementation. Multienzymes at different doses did not significantly affect the digestibility of nutrients, blood serum biochemical constituent, inner body organs, and markers of functions of liver and renal organs. In conclusion, the highest BWG and the best FCR and EPI for the whole period were from broilers given continuous 1 and 1.5 mL/L drinking water or intermittent multienzyme supplementation at 1.5 mL/L drinking water. Furthermore, intermittent supplementation of enzymes at 1 mL/L drinking water and continuous supplementation at 0.5 mL/L drinking water can be investigated in further experiments as a tool to improve broiler growth performance and economic traits and to decrease the cost of enzyme application.

Objectives: To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.Results: Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.Conclusion: This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of soybean meal and multienzyme supplementation on intestinal histopathology of beluga sturgeon ( Huso huso). Nine diets were formulated (40% crude protein, 13% crude fat and 4150 kcal/kg gross energy) in which fish meal was replaced by soybean meal at 0, 40 and 80% supplemented or not with 1 or 2 g/kg of multienzyme Kemin. Fish (initial weight of 820 g) were fed on experimental diets four times daily at 1% body weight for 12 weeks. From histological analyses, intact villus with normal mucosa was found in the FM group, representing the presence of intact epithelium. Supplementation of enzyme at both levels and soybean meal (alone) in the diet resulted in histopathological alterations in the intestine including mild immune cells infiltration, villus blunting, villus fusion, mononuclear immune cells infiltration as well as necrosis. Soybean meal decreased the length of villuses in the proximal and distal intestines ( P < 0.05). Enzyme was not able to ameliorate the derangements induced by 80% replacement level of fish meal with soybean meal (treatments SB80E1% and SB80E2%). However, co-administrating of the enzyme at both levels with 40% soybean meal (treatments SB40E1% and SB40E2%) was able to inhibit the soybean meal-induced histopathological impairments. [ABSTRACT FROM AUTHOR]

The current study investigated the effects of dietary protein levels and multienzyme supplementation on the growth performance, subclinical necrotic enteritis, intestinal mucosal barrier and microflora of broilers fed diets containing meals of cottonseeds, rapeseeds, peanuts and sesame. A total of 480 one-day-old male Cobb broilers were assigned to a 2 × 2 factorial arrangement of treatments with 6 replicated pens of 20 birds each. Experimental factors included two dietary protein levels at 205 or 225 g/kg, and without or with multienzymes. The multienzymes contained phytase, protease and xylanase at 1000, 2000 and 2000 U/kg of feed, respectively. The trial lasted for 21 days. The high protein diet increased ( P < 0.05) small intestinal lesions, duodenal mucin 2 secretion, ileal counts of C. perfringens and E. coli , but decreased ( P < 0.05) secretary IgA (sIgA), polymeric Ig receptor (pIgR), ileal counts of Lactobacilli and Bifidobacteria . Multienzymes increased ( P < 0.05) feed intake, body weight gain, pIgR, sIgA, and ileal counts of Lactobacilli and Bifidobacteria and decreased ( P < 0.05) intestinal lesions, serum a-toxin antibodies, mucin 2 expression and ileal count of C. perfringens , but the magnitude of the effect of multienzymes was dependent on the protein level in the diet (interaction, P < 0.05). It can be concluded that diets containing high nonconventional protein can increase the incidence of subclinical necrotic enteritis, while supplementing multienzymes can decline this risk in broilers. [ABSTRACT FROM AUTHOR]

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans -sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives. [ABSTRACT FROM AUTHOR]

O -Acetylation of sialic acid (Sia) modulates its recognition by sialic acid-binding proteins and plays an important role in biological and pathological processes. 9-O-Acetylation is the most common modification of sialic acid in human. However, study of O -acetylated sialoglycans is hampered due to the instability of O -acetyl group towards pH changes and sensitivity to esterases. Our previous studies demonstrated a chemical biology method to this problem by replacing the oxygen atom in the C9 ester group of sialic acid by a nitrogen to form an amide. Here, we synthesized a library of sixteen new 9-acetamido-9-deoxy- N -acetylneuraminic acid (Neu5Ac9NAc)-containing α2–3- and α2–6-linked sialosides with various underlying glycans using efficient one-pot three-enzyme (OP3E) sialylation systems. Neu5Ac9NAc-containing compounds with a para -nitrophenol aglycon have been used together with their 9- O -acetyl analogs in microtiter plate-based high-throughput substrate specificity studies of nine different sialidases including those from humans and bacteria. In general, similar to 9-O-acetylation, 9- N -acetyl modification of sialic acid in the substrates lowers sialic acid-cleavage activity of most sialidases. In most cases, Neu5Ac9NAc is a good analog of 9- O -acetyl sialic acid. However, exceptions do exist. For example, 9- N - and 9- O -acetyl modifications have different effects on the sialosides cleave efficiencies of a commercially available C. perfringens sialidase as well as recombinant Streptococcus pneumoniae sialidase SpNanC and Bifidobacterium infantis sialidase BiNanH2. The mechanism for the difference awaits further investigation. [ABSTRACT FROM AUTHOR]

Helicobacter pyloriα1–3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L−1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of l-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT). [ABSTRACT FROM AUTHOR]

An experiment was conducted to evaluate the effects of dietary inclusion of (multi-enzymes) maxi-zyme at different energy and protein level on performance of egg-type chicks during hot-dry summer. After feeding a standard diet from 0-21d of age, the chicks were randomly distributed into four dietary treatment groups viz., T1 with high energy 2800 kcal ME kg-1 and high protein 18.0% (HEHP), T2 HEHP diet with maxi-zyme 0.025% and T3 with low energy 2700 kcal ME kg-1 low protein 17.34% (LELP) and T4 with LELP diet with maxi-zyme 0.025%. Each treatment is with fifty birds divided in five replicated of ten birds each. Experiment was carried out during hot-dry summer (April-May, 28.0±0.12 oC to 35.25±0.37 oC and RH %: 68.95±0.90 to 79.15±0.61). Production performance, immune organ weight, blood biochemical and intestinal morphometry were measured at 42nd and 63rd day post hatch. Body weight gain, Feed intake and FCR improved significantly (P<0.05) in HEHP+maxi-zyme group. H:L ratio significantly (p<0.05) reduced in maxi-zyme supplemented group at 42nd as well as 63rd day of age. Immune organ like thymus, spleen and bursa had significantly (p<0.05) higher weight in maxi-zyme supplemented group. Total protein (p<0.001), SGOT (p<0.001), SGPT (p<0.001), creatinine (p<0.05) were significantly improved while cholesterol, uric acid & ALP was significantly (p<0.001) reduced in maxi-zyme supplemented diets. Villus height & crypt depth improved significantly (p<0.001) in maxizyme supplemented group at 42nd as well as 63rd day of age. Thus, it was concluded that high-energy (2800 kcal ME kg-1) and high protein (18% CP) diet was beneficial for egg type starting chicks during dry summer. Moreover, the inclusion of maxi-zyme @ 0.025% gave further advantage to improve performance and reduce thermal stress. [ABSTRACT FROM AUTHOR]

This study is aimed at determining the effects of simple and cheap hydrothermal techniques on the antinutrtional components and in vitro multienzymes protein digestibility of Vigna racemosa, an underutilised hard-to-cook legume. Seeds of V. racemosa were subjected to aqueous soaking and four hydrothermal processing methods - atmospheric boiling and steaming; pressure boiling and steaming. The effects of soaking and the hydrothermal techniques on the antinutrtional components (phytic acid, saponin, tannin and trypsin inhibitor activity) and the in vitro protein digestibility were investigated. Increase in hydration level caused more seepage of the antinutrients into the soaking water. With the exception of trypsin inhibitor activity that was totally eliminated, other antinutritonal components experienced significant reduction (p<0.05) when the seeds were subjected to the various hydrothermal processing techniques. The raw sample has protein digestibility of 40.11% while steaming at atmospheric pressure and steaming at elevated pressure caused percentage digestibility of 87.11% and 87.36%, respectively. The boiling treatments resulted in better percentage protein digestibility of 87.91%. Application of these simple and cost effective hydrothermal processing methods resulted in significant decrease in the antinutrtional components and hence improvement of protein digestibility. Increase in utilisation of this legume for foods could strengthen dietary diversity and healthy eating habit and thus alleviate the problems of protein energy malnutrition and food insecurity. [ABSTRACT FROM AUTHOR]

The long-term excessive use of chlorothalonil (CTN) can lead to serious environmental pollution, which presents a cause for concern. Here, we investigated the degradation of CTN by using an effective strain, TUAH-1, which was identified as Enterobacter cloacae according to the morphological approach, 16S rDNA gene sequence and phylogenetic tree analysis. Treatment with TUAH-1 demonstrated that the maximum processing capability was 74 mg CTN per gram dry cells in 48 h under optimum conditions (30 °C–35 °C, pH 7.0). After treatment with 21.3 mg dry cells, the degradation efficiency was 97.4% for 20 mg l -1 CTN in the aqueous phase. Meanwhile, treatment with crude enzyme solution containing 2.0 mg ml -1 protein led to complete degradation of 20.0 mg l -1 CTN within 10 h, which is significantly faster than that of TUAH-1. The results also showed that CTN could not be detected after treatment with 5.68 g TUAH-1 per kg soil for 48 h when the original CTN content in the soil was 10 mg kg -1 . After treatment with THAH-1 or its crude proteins, many degradation products were detected by HPLC-MS, suggesting two possible degradation pathways. The mechanism of CTN degradation by TUAH-1 was noted to be multienzymatic catalysis comprising glyceraldehyde-3-phosphate dehydrogenase and glutathione S-transferase. [ABSTRACT FROM AUTHOR]

In this study, it was aimed to determine the performances and some carcass and small intestine properties of broiler chickens fed diets in which sunflower meal (SFM) replaced soybean meal (SBM) and supplemented or not with multienzyme. With this aim, a total of 450 1-day-old age mixed-sex Ross 308 broiler chicks were used in a completely randomized design, with five treatments and three replicates of thirty birds each. The experimental diets consisted of a control group and two levels of SFM with or without enzyme replacing 50 or 100% of SBM protein, respectively. Hence, treatment groups were: control (100SBM), 50SFM, 100SFM, 50SFME, and 100SFME). Feed and fresh water was given freely. At the end of the experiment (42 days of age), 100SBM broilers had higher body weight and cold carcass weight than all other groups (P <0.05) and had lower feed conversion ratio (FCR) compared to 50SFME broilers (P <0.05). Feed consumption, weights of hearth, liver, gizzard, abdominal fat and small intestine and length of small intestine were not found different among the groups (P>0.05). Our observations demostrated that addition of the 0.05% multi-enzyme mixture into SFM diets of broiler chickens had not positive impact on the availability of SFM and broiler performance. [ABSTRACT FROM AUTHOR]

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σI factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum. In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σI factors and by the vegetative σA factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σIand σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σA promoter. With the exception of C. cellulovorans, both σI and σA promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components. IMPORTANCE: Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass. [ABSTRACT FROM AUTHOR]

A novel ECL biosensor for cytosensing and cell surface carbohydrate expression evaluation was developed, by the integration of the peptide modified interface for highly specific carbohydrate recognition and sodium alginate loaded glucose oxidase as the signal probe with high signal amplification efficiency. A cysteine-terminated peptide self-assembled on the electrode through Au–S bond to construct a functional interface for cell capture, with decent biocompatibility and high affinity for the human breast cancer cell MCF-7. Concanavalin A lectin modified gold nanoparticles specifically recognized the cell surface carbohydrates and were absorbed on the electrode, followed by the immobilization of multiple glucose oxidase conjugated sodium alginate, which could remarkably increase the sensitivity of the biosensor with enhanced catalysis. The as-proposed ECL cytosensor was successfully applied for the detection of the MCF-7 tumor cells, whose glycans on the cell membranes are over-expressed. A low detection limit of 150 cells mL −1 was obtained, with a wide dynamic linear range from 5.0×10 2 to 5.0×10 5 cells mL −1 . Due to the excellent sensitivity, stability and biocompatibility, the ECL biosensor would be promising in reliable diagnostics of glycan relevant biomarkers for cancer and other diseases. [ABSTRACT FROM AUTHOR]

We earlier identified Bacillus subtilis NG105 and Trichoderma asperelllum NG125 isolated from fruit and vegetable wastes (FVWs) and established that multienzyme activity viz pectinase, cellulase and amylase (PC&A) activity was localized exclusively in cell exudates (CEs). In this study, we evaluated the best rotten or leftover FVWs of Bael, Guava, Mosambi, Beetroot, Banana, Aonla, Mango and Cabbage as potential substrates under solid-state fermentation (SSF) for isolated microbial strains to multi-enzyme production. The results showed that after SSF, the multienzyme activity of microbial CEs of both NG105 (394.6, 296.1, 341.4 μmol ml−1min−1 of PC&A respectively) and NG125 (420.5, 347.3, 330.7 μmol ml−1min−1 of PC&A respectively) extracted from mosambi peel performed best. The multienzyme present in CEs was further partially purified using five different chemical solvents; acetone, trichloroacetic acid, ammonium sulphate (70%), ethanol, and n-propanol. Results revealed that partially purified multienzyme (PPM) of both NG105 (415.2, 425.4, 414.3 μmol ml−1min−1 of PCA respectively) and NG125 strain (528.5, 509.2, 467.6 μmol ml−1min−1 of PC&A respectively) found to be highest in PPM extracted with acetone. The result indicates that mosambi peel may be used as the best FVWs as the substrate for our isolated strains for multienzyme production and the acetone purification method was well suited for the enhancement of the enzymatic activity. Moreover, the produced multienzyme (in CEs form) either in the system in which they are produced (eg. juice clarification) or extracted and partially purified form may be further utilized for other purposes (eg. food, animal feed, bio-fuel, detergent, industries etc). • Precise utilization of FVWs can reduce the harmful environmental impact. • Mosambi peel showed the best multi-enzyme activity among all tested FVWs. • The multienzyme extraction through acetone was best over other methods. • Mosambi peel can be used as cheap substrate for microbial mutli-enzyme production. [ABSTRACT FROM AUTHOR]

We report on the isolation and characterization of an acid- and bile-tolerant bacterial strain, Bacillus sp. YB1701 with antibacterial and quorum-quenching activity. Strain YB1701 was isolated from coastal sediment samples and characterized by biochemical tests and 16S rRNA sequencing. In vitro study indicated that strain YB1701 can survive at pH 2.0 for up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. Strain YB1701 showed antimicrobial activity against fish pathogens Aeromonas hydrophila and Vibrio parahemolyticus using an agar well diffusion assay. The trial test showed dietary supplementation of YB1701 significantly improved the resistance of Carassius auratus gibelio against A. hydrophila challenge. The safety assessment revealed that the isolate Bacillus sp. YB1701 was not cytotoxic to Carassius auratus gibelio or mice and did not exhibit hemolytic activity on rabbit blood agar plate. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that YB1701 was susceptible to selected antibiotics. Under laboratory conditions, the degradation rate of organic waste (predominately fish excrement) for 14 days by YB1701 was 79.69%. Results from the present study suggest that strain YB1701 is a potential probiotic strain and can be used in aquaculture for degrading organic waste and improving disease resistance of fish against bacterial infection. Further study is needed to assess the utility of strain YB1701 on a commercial scale.

The synergistic combination of current biotechnological and nanotechnological research has turned to multienzyme co-immobilization as a promising concept to design biocatalysis engineering. It has also intensified the development and deployment of multipurpose biocatalysts, for instance, multienzyme co-immobilized constructs, via biocatalysis/protein engineering to scale-up and fulfil the ever-increasing industrial demands. Considering the characteristic features of both the loaded multienzymes and nanostructure carriers, i.e., selectivity, specificity, stability, resistivity, induce activity, reaction efficacy, multi-usability, high catalytic turnover, optimal yield, ease in recovery, and cost-effectiveness, multienzyme-based green biocatalysts have become a powerful norm in biocatalysis/protein engineering sectors. In this context, the current state-of-the-art in enzyme engineering with a synergistic combination of nanotechnology, at large, and nanomaterials, in particular, are significantly contributing and providing robust tools to engineer and/or tailor enzymes to fulfil the growing catalytic and contemporary industrial needs. Considering the above critics and unique structural, physicochemical, and functional attributes, herein, we spotlight important aspects spanning across prospective nano-carriers for multienzyme co-immobilization. Further, this work comprehensively discuss the current advances in deploying multienzyme-based cascade reactions in numerous sectors, including environmental remediation and protection, drug delivery systems (DDS), biofuel cells development and energy production, bio-electroanalytical devices (biosensors), therapeutical, nutraceutical, cosmeceutical, and pharmaceutical oriented applications. In conclusion, the continuous developments in nano-assembling the multienzyme loaded co-immobilized nanostructure carriers would be a unique way that could act as a core of modern biotechnological research. [Display omitted] • Immobilized multienzymatic systems represent a versatile platform for biocatalysis. • Prospective nano-carriers as host for immobilized multienzymatic systems are reviewed. • Biotechnological applications of immobilized multienzymatic systems are outlined. • Improvement of stability and reusability of the co-immobilized enzymes is emphasized. • Future trends for widespread applications of multienzymatic systems are highlighted. [ABSTRACT FROM AUTHOR]

The enzymatic synthesis of biologically important and structurally unique human P1PK blood group type P1 pentasaccharide antigen is described. This synthesis features a three-step sequential one-pot multienzyme (OPME) glycosylation for the stepwise enzymatic chain elongation of readily available lactoside acceptor with cheap and commercially available galactose and N -acetylglucosamine as donor precursors. This enzymatic synthesis provides an operationally simple approach to access P1 pentasaccharide and its structurally related Gb3 and P1 trisaccharide epitopes. [ABSTRACT FROM AUTHOR]

The controlled spatial organization or compartmentalization of multi-enzyme cascade reactions to transfer a substrate from one enzyme to another for substrate channeling on scaffolds has sparked increasing interest in recent years. Here, we use graphene oxides to study the dependence of the activity of cascade reactions in a closely packed, randomly immobilized enzyme system on a 2 D scaffold. We first observe that the hydrophobicity of graphene oxides and various enzyme architectures for co-immobilized systems are important attributes for achieving high product-conversion rates. A transient time close to 0 s can be achieved if enzymes are randomly immobilized close to one another, owing to direct molecular channeling. This contributes to overcoming complications regarding control of the spatial arrangement of the enzymes. Furthermore, a fabricated bienzyme paper can be used for glucose detection with high stability, reusability, and enhanced substrate channeling. Our findings provide new guidance for enzyme orientation on 2 D scaffolds, which may be extrapolated to other multienzyme cascade systems. [ABSTRACT FROM AUTHOR]

The present paper studied the effect of screened wheat (SW) levels and a multienzyme diet on Japanese quail performance, intestinal performance, and microbial population. A total of 480 unsexed 10-day-old quail chicks were assigned to a multienzyme diet (0 or 0.5 g/kg) and SW (0%, 5%, 10%, or 15%) in a 2 × 4 factorial arrangement of treatments with 4 replications and 15 quails in each in a completely randomized design. No differences were observed in performance between diets at 10 to 36 days of age. Increases in dietary SW levels decreased villus height, villus width, villus surface, and height:crypt depth ratio and increased crypt depth (linear; P < 0.01). Increasing SW levels in diets decreased the number of lactobacilli of the cecum and ileum (linear; P < 0.01), while it increased the number of Enterobacteriaceae in the cecum and ileum (quadratic; P < 0.01). Increasing SW level in diets decreased the population of lactobacilli, increased Enterobacteriaceae, and reduced intestinal morphology growth. Protein synthesis and DNA contents of the jejunal mucosa were decreased linearly with increasing levels of SW in diets (P < 0.05). Finally, it was concluded that inclusion of SW up to 15% had no adverse effect on performance, molecular, and intestinal morphological parameters of Japanese quail. [ABSTRACT FROM AUTHOR]

Biomimetic synthesis routes towards the important natural d-mannosyl donor guanosine 5′-diphospho- d-mannose (GDP-Man) rely on kinase-catalyzed nucleotide triphosphate (NTP)-dependent phosphorylations of d-mannose (Man), to give d-mannose 6-phosphate or α- d-mannose 1-phosphate (αMan 1-P) as an intermediate product. A GDP-Man synthesis not requiring the kinase/NTP system would be practical and cost-effective. Here, we have developed a multienzyme cascade towards GDP-Man, characterized in that αMan 1-P was obtained by a diastereoselective phosphatase-catalyzed phosphorylation of Man. α- d-Glucose 1-phosphate (αGlc 1-P), prepared in situ through phosphorylase-catalyzed conversion of sucrose in the presence of inorganic phosphate, was used as an expedient phosphoryl donor. The incipient αMan 1-P and guanosine triphosphate (GTP) were converted into GDP-Man by a highly manno compared to gluco selective nucleotidyltransferase. Pyrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reaction towards GDP-Man. The enzymatic cascade was operated with the αMan 1-P and the GDP-Man formation decoupled from one another ( sequential mode) or having all steps run concurrently ( simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate αMan 1-P in simultaneous-mode reactions was important to promote phosphorylation of Man from αGlc 1-P in high efficiency, avoiding loss of sugar 1-phosphates by hydrolysis. Under optimized conditions for the one-pot transformation involving four enzymes, 100 mM (67 g L−1) GDP-Man was prepared from 140 mM sucrose and phosphate, using 400 mM Man as the phosphoryl acceptor. The product was recovered by anion-exchange and size-exclusion chromatography in ≥95% purity in about 50% yield (100 mg). These results demonstrate for the first time the practical use of a phosphorylase-phosphatase combi-catalyst as an alternative to the canonical kinase for the anomeric phosphorylation of the sugar substrate in nucleoside diphospho-sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate αGlc 1-P rather than from NTP, particularly GTP, appears advantageous specifically in cases where the sugar acceptor is a bulk commodity that can be applied in suitable excess to the phosphatase reaction. [ABSTRACT FROM AUTHOR]

The present study was conducted to assess the effect of supplementation of Multi-enzymes with lysophospholipids on production performance of pure line White Leghorn layers. Body weights before and after the experiment did not differ significantly across the experimental diets. Irrespective of the dietary treatments, the birds gained 3.83 per cent of live weight relative to its initial body weight. Mean per cent Hen housed egg production was significantly (P<0.05) higher in diet supplemented with 0.10 MEC-L than other groups. Mean egg weight and average daily feed consumption during 25 to 35 weeks of age indicated no significant effect of enzyme supplementation. Average daily feed consumption per bird in control, 0.05 % and 0.1% multi-enzyme supplemented groups was 108.13, 105.66 and 107.67 g respectively and birds offered control diet recorded numerically more feed intake than enzyme supplemented groups. Comparatively low feed per egg was observed in 0.10 per cent group followed by 0.05 per cent group which offers economic benefits than control diets. However, the egg quality traits between different dietary enzyme supplementation groups showed no significant difference. [ABSTRACT FROM AUTHOR]

We present the second-generation GeomBD Brownian dynamics software for determining interenzyme intermediate transfer rates and substrate association rates in biomolecular complexes. Substrate and intermediate association rates for a series of enzymes or biomolecules can be compared between the freely diffusing disorganized configuration and various colocalized or complexed arrangements for kinetic investigation of enhanced intermediate transfer. In addition, enzyme engineering techniques, such as synthetic protein conjugation, can be computationally modeled and analyzed to better understand changes in substrate association relative to native enzymes. Tools are provided to determine nonspecific ligand-receptor association residence times, and to visualize common sites of nonspecific association of substrates on receptor surfaces. To demonstrate features of the software, interenzyme intermediate substrate transfer rate constants are calculated and compared for all-atom models of DNA origami scaffold-bound bienzyme systems of glucose oxidase and horseradish peroxidase. Also, a DNA conjugated horseradish peroxidase enzyme was analyzed for its propensity to increase substrate association rates and substrate local residence times relative to the unmodified enzyme. We also demonstrate the rapid determination and visualization of common sites of nonspecific ligand-receptor association by using HIV-1 protease and an inhibitor, XK263. GeomBD2 accelerates simulations by precomputing van der Waals potential energy grids and electrostatic potential grid maps, and has a flexible and extensible support for all-atom and coarse-grained force fields. Simulation software is written in C++ and utilizes modern parallelization techniques for potential grid preparation and Brownian dynamics simulation processes. Analysis scripts, written in the Python scripting language, are provided for quantitative simulation analysis. GeomBD2 is applicable to the fields of biophysics, bioengineering, and enzymology in both predictive and explanatory roles. [ABSTRACT FROM AUTHOR]

Summary Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes. [ABSTRACT FROM AUTHOR]

BACKGROUND Four enzymes were immobilized for the production of optically pure natural and non-natural L-amino acids via the 'Double-Racemase Hydantoinase Process'. Immobilization constitutes an empirical process, and for each enzyme we tested 11 carriers with different functional groups; epoxide, EC-EP, EC-HFA, IB-150 and IB-350, carboxylic acid IB-C435, quaternary ammonium IB-A161, IB-A171 and IB-A369, aromatic group IB-S861, and hydroxyl group IB-S60S and IB-S60P. RESULTS Each protein showed preference for binding on one or several supports: D,L-hydantoinase/ IB-350, hydantoin racemase/ EC-EP, L-carbamoylase/ EC-EP and carbamoyl racemase/ IB-A161. The process was optimized for each enzyme by modifying temperature, pH and ionic strength. For the enzymatic cascade, it was demonstrated that it was essential to use supports having homogeneous characteristics. The product of the first reaction is the substrate of the next one, and so on. Free mass diffusion from one enzyme to the other is crucial to avoid retention on the support and to maintain the protein-substrate interaction constant. CONCLUSION It was proved that support size, weight and hydrophobicity must be homogeneous to avoid the formation of separate layers of the matrix selected. For this reason, the support IB-350 was used for the four enzymes, even though it may not be the most efficient one for some of them. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD+ substrate channeling by specifically shifting NAD+ between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) show great potential for disease diagnostics due to their specific molecular profiles. Detection of miRNAs remains challenging and often requires sophisticated platforms. Here we report a multienzyme-functionalized magnetic microcarriers-assisted isothermal strand-displacement polymerase reaction (ISDPR) for quantitative detection of miRNAs. Magnetic micro-carriers (MMCs) were functionalized with molecular beacons to enable miRNAs recognition and magnetic separation. The target miRNAs triggered a phi29-mediated ISDPR, which can produce biotin-modified sequences on the MMCs. Streptavidin–alkaline phosphatase was then conjugated to the MMC surface through biotin–streptavidin interactions. In the presence of 2-phospho- L -ascorbic acid, miRNAs were quantitatively determined on a screen-printed carbon electrode from the anodic current of the enzymatic product. We show that this method enables detection of miRNAs as low as 9 fM and allows the discrimination of one base mismatched sequence. The proposed method was also successfully applied to analyze miRNAs in clinical tumor samples. This paper reports a new strategy for miRNAs analysis with high sensitivity, simplicity, and low cost. It would be particularly useful for rapid point-of-care testing of miRNAs in clinical laboratory. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli . ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160 min, ALA concentration was increased to 5.4 mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production. [ABSTRACT FROM AUTHOR]

Filter aided sample preparation (FASP) and related methods gain increasing popularity for proteomic sample preparation. Nevertheless, the originally published FASP method has been criticized by several authors, who reported low digestion performance. In this work, we re-evaluate FASP and the related multienzyme digestion (MED) FASP method. We use different types of animal tissues and cultured cells and test the performance of the method under various conditions. We analyze the protein to peptide conversion by assessing the yield of peptides, frequency of peptides with missed cleavage sites, and the reproducibility of FASP. We identify conditions allowing efficient protein processing with high peptide yields and demonstrate advantages of the two step digestion strategy over single step digestion with trypsin. In addition, we show that FASP outperforms in-solution cleavage strategies. Our results clearly demonstrate that the performance of digestion varies between different types of samples. We show that MED FASP in combination with the total protein approach provides highly reproducible protein abundance values. The presented data can be used as a guide for optimization of sample processing. [ABSTRACT FROM AUTHOR]