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The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL−1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg−1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7–9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

β-1,3-1,4-Glucanases are a type of hydrolytic enzymes capable of catalyzing the strict cleavage of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in β-D-glucans and have exhibited great potential in food and feed industrials. In this study, a novel glycoside hydrolase (GH) family 12 β-1,3-1,4-glucanase (CtGlu12A) from the thermophilic fungus Chaetomium sp. CQ31 was identified and biochemically characterized. CtGlu12A was most active at pH 7.5 and 65 °C, respectively, and exhibited a high specific activity of 999.9 U mg−1 towards lichenin. It maintained more than 80% of its initial activity in a wide pH range of 5.0–11.0, and up to 60 °C after incubation at 55 °C for 60 min. Moreover, the crystal structures of CtGlu12A with gentiobiose and tetrasccharide were resolved. CtGlu12A had a β-jellyroll fold, and performed retaining mechanism with two glutamic acids severing as the catalytic residues. In the complex structure, cellobiose molecule showed two binding modes, occupying subsites −2 to −1 and subsites + 1 to + 2, respectively. The concave cleft made mixed β-1,3-1,4-glucan substrates maintain a bent conformation to fit into the active site. Overall, this study is not only helpful for the understanding of the substrate-binding model and catalytic mechanism of GH 12 β-1,3-1,4-glucanases, but also provides a basis for further enzymatic engineering of β-1,3-1,4-glucanases.

The effects of Konjac mannan oligosaccharides (KMOS) on diphenoxylate-induced constipation in mice were investigated in the present study. After administration with KMOS at 1800 mg/kg·bw, the weight and number of black feces within 24 h, and the gastrointestinal transit ratio were distinctly increased by 96.8%, 156.5% and 45.3%, respectively. Furthermore, KMOS regulated the levels of enteric nervous system parameters, including motilin, somatostatin, substance P, acetyl cholinesterase, endothelin and vasoactive intestinal peptide. Additionally, KMOS could recover the characteristics of small intestine villi and inhibit the reduction of mucosal thickness. KMOS administration produced more short chain fatty acids and lactic acid compared with the control group. The availability of 5-hydroxytryptamine and the expression of serotonin transporter were ameliorated in the KMOS treatment groups. Collectively, these findings suggested that KMOS could be considered as a functional food material for regulating intestinal function and alleviating constipation.

D-Mannose isomerase can reversibly catalyze D-fructose to D-mannose which has various beneficial effects. A novel D-mannose isomerase gene (PsMIaseA) from Pseudomonas syringae was cloned and expressed in Escherichia coli. The recombinant D-mannose isomerase (PsMIaseA) showed the highest amino acid sequence homogeneity of 50% with ManI from Thermobifda fusca. PsMIaseA was purified through Ni-NTA chromatography, and its specific activity was 818.6 U mg-1. The optimal pH and temperature of PsMIaseA were pH 7.5 and 45 °C, respectively. The enzyme was stable within a wide pH range from 5.0 to 10.0. It could efficiently convert D-fructose to D-mannose without any metal ions. When PsMIaseA was incubated with 600 g/L D-fructose for 6 h, the space-time yield of D-mannose reached 27.2 g L-1 h-1 with a maximum conversion ratio of 27%. Therefore, the D-mannose isomerase may be suitable for green production of D-mannose.

The problem of stochastic adaptive air-fuel ratio control by the dynamic model of biogas-fuelled engines is investigated in this paper. An adaptive law is employed to estimate the theoretical air-fuel ratio, which is undetermined due to the uncertainty of the methane concentration in the biogas. A stochastic adaptive air-fuel ratio controller in consideration of the stochasticity of the residual gas is designed based on the adaptive law, and the closed-loop system is proven to be mean-square stable. The proposed stochastic adaptive air-fuel ratio controller is validated through a numerical simulation when the theoretical air-fuel ratio is unknown constants and jump signals.

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. β-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-β-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A–M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (−3 to +4) in the catalytic groove of RmMan134A. Mannose in the −1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A–M5 indicated that subsites −2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A–M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 β-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel β-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization

Background β-Mannanase randomly cleaves the β-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of β-mannanase have tremendously expanded in recent years, the wild-type β-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 β-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited. Results To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated β-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (k cat/K m) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A. Conclusions Through directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure–function relationship of GH family 5 β-mannanase was acquired, which could be used for modifying β-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a β-mannanase from zygomycete engineered by directed evolution. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0833-x) contains supplementary material, which is available to authorized users.

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 β-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL−1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50 °C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1–F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α- d -galactosyl-β- d -mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2–3 rather than those with DP 4–5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.

Natural polysaccharides, particularly galactomannans, are potential candidates for treatment of alcoholic liver diseases (ALD). However, applications are restricted due to the physicochemical properties associated with the high molecular weight. In this work, guar gum galactomannans were partially hydrolyzed by &beta, mannanase, and the molecular mechanisms of hepatoprotective effects were elucidated both in vitro and in vivo. Release of lactate dehydrogenase and cytochrome C were attenuated by partially hydrolyzed guar gum (PHGG) in HepG2 cells, due to protected cell and mitochondrial membrane integrity. PHGG co-administration decreased serum amino transaminases and cholinesterase levels of acute alcohol intoxicated mice, while hepatic pathologic morphology was depleted. Activity of superoxide dismutase, catalase, and glutathione peroxidase was recovered to 198.2, 34.5, 236.0 U/mg protein, respectively, while malondialdehyde level was decreased by 76.3% (PHGG, 1000 mg/kg∙day). Co-administration of PHGG induced a 4.4-fold increment of p-AMPK expression, and lipid metabolism was mediated. PHGG alleviated toll-like-receptor-4-mediated inflammation via the signaling cascade of MyD88 and I&kappa, B&alpha, decreasing cytokine production. Moreover, mediated expression of Bcl-2 and Bax was responsible for inhibited acute alcohol-induced apoptosis with suppressed cleavage of caspase 3 and PARP. Findings gained suggest that PHGG can be used as functional food supplement for the treatment of acute alcohol-induced liver injury.

A novel glycoside hydrolase family 5 β-mannosidase gene (AcMan5) from the filamentous fungus Absidia corymbifera D1 was cloned and expressed in Escherichia coli. AcMan5 had an open reading frame of 1311 bp encoding 436 amino acids. The β-mannosidase (AcMan5) shared highest amino acid sequence identity (73%) with the β-mannosidase from Rhizomucor miehei. The purified β-mannosidase was a monomer of 51.9 kDa. Its optimal pH and temperature were pH 5.5 and 60 °C, respectively. The enzyme was stable up to 60 °C and within a pH range of 4.0⿿10.5. It was found to be a typical exo-β-mannosidase acting on the non-reducing end of manno-oligosaccharides. In addition, AcMan5 displayed high transglycosylation activity. It could transfer mannose residue to six acceptors ⿿ glucose, galactose, fructose, gentiobiose, laminaribiose and sucrose ⿿ among 20 candidates at low donor concentration in a short time. Due to its hydrolytic and transglycosylation properties, AcMan5 may have potential in several industrial applications. This is the first report on a β-mannosidase from Absidia species.

Hyperspectral imaging at visible and short near infrared (VIS/SNIR) region has been used to estimate the pigment content of leaves. A complicating feature of measurements with any hyperspectral imaging methodology is the large amount of information generated during the measurement process. In this paper we discuss the identification of the desirable information using independent component analysis (ICA). After hyperspectral image acquisition and pre-processing, the average spectra obtained from the region of interest (ROI) in cucumber leaves were used for model development. Additionally a multi-linear regression model was developed for the prediction of cucumber leaf chlorophyll content. When compared with normal principal component analysis (PCA), the ICA multi-linear regression model provided improved estimates. When the calibration models were applied to an independent validation set, chlorophyll content was reasonably well predicted with a high correlation (r2 = 0.774). Depending on the sample, the technique enabled the identification and characterization of the relative content of various chlorophyll types that were distributed within the cucumber leaves. Typically low levels of chlorophyll at leaf margins and higher levels along main vein regions were identified. Our results indicate that hyperspectral imaging exhibits considerable promise for predicting pigments within cucumber leaves and furthermore can be applied non-destructively and in situ to living plant samples.

A new room temperature gas sensor was fabricated with pigment-sensitized TiO2 thin film as the sensing layer. Four natural pigments were extracted from spinach (Spinacia oleracea), red radish (Raphanus sativus L), winter jasmine (Jasminum nudiflorum), and black rice (Oryza sativa L. indica) by ethanol. Natural pigment-sensitized TiO2 sensor was prepared by immersing porous TiO2 films in an ethanol solution containing a natural pigment for 24h. The hybrid organic-inorganic formed films here were firstly exposed to atmospheres containing methylamine vapours with concentrations over the range 2-10 ppm at room temperature. The films sensitized by the pigments from black-rice showed an excellent gas-sensitivity to methylamine among the four natural pigments sensitized films due to the anthocyanins. The relative change resistance, S, of the films increased almost linearly with increasing concentrations of methylamine (r=0.931). At last, the black rice pigment sensitized TiO2 thin film was used to determine the biogenic amines generated by pork during storage. The developed films had good sensitivity to analogous gases such as putrscine, and cadaverine that will increase during storage.

A thermostable xylanase (McXyn10) from the thermophilic fungus Malbranchea cinnamomea strain S168 was purified and biochemically characterized. The enzyme was purified to homogeneity with a molecular mass of 43.5 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme were pH 6.5 and 80°C, respectively. The enzyme showed a broad range of pH stability (pH 4.0-10.5), and was stable up to 70°C with a thermal denaturing half life of 76.0 min. The enzyme exhibited strict specificity for various xylans as substrates, but displayed no activity toward other tested polysaccharides. McXyn10 hydrolyzed birchwood xylan, beechwood xylan and oat-spelt xylan, yielded mainly xylobiose, xylotriose and xylooligosaccharides with degree of polymerization (DP) above 5, while yielded xylobiose from xylotriose and xylotetraose. The xylanase gene was further cloned. It had an open reading frame of 1191 bp with two introns. The deduced amino acid sequence of the gene showed highest identity (58%) with a glycoside hydrolase family 10 xylanase from Aureobasidium pullulans.

Design and fabrication of an ammonia sensor operating at room temperature based on pigment-sensitized TiO2 films was described. TiO2 was prepared by sol–gel method and deposited on glass slides containing gold electrodes. Then, the film immersed in a 2.5 × 10−4 M ethanol solution of cyanidin to absorb the pigment. The hybrid organic–inorganic formed film here can detect ammonia reversibly at room temperature. The relative change resistance of the films at a potential difference of 1.5 V is determined when the films are exposed to atmospheres containing ammonia vapors with concentrations over the range 10–50 ppm. The relative change resistance, S, of the films increased almost linearly with increasing concentrations of ammonia (r = 0.92). The response time to increasing concentrations of the ammonia is about 180–220 s, and the corresponding values for decreasing concentrations 240–270 s. At low humidity, ammonia could be ionized by the cyanidin on the TiO2 film and thereby decrease in the proton concentration at the surface. Consequently, more positively charged holes at the surface of the TiO2 have to be extracted to neutralize the adsorbed cyanidin and water film. The resistance response to ammonia of the sensors was nearly independent on temperature from 10 to 50 °C. These results are not actually as good as those reported in the literature, but this preliminary work proposes simpler and cheaper processes to realize NH3 sensor for room temperature applications.

The objective of this study was to investigate the spectral behavior of the relationship between reflectance and chlorophyll content and to develop a technique for non-destructive chlorophyll estimation and distribution in leaves using hyperspectral imaging. The hyperspectral imaging data cube of cucumber (Cucumis sativus) leaves in the range of 450-850 nm was investigated and preprocessed. Sixty optical signatures or indices as a function of the associated reflectance (R(λ)) at the special wavelength (λ) nm which proposed in the literatures were used to predict the total chlorophyll content in cucumber leaves. Finally, R(710)/R(760), (R(780)-R(710))/(R(780)-R(680)), (R(750)-R(705))/(R(750)+R(705)), (R(680)-R(430))/(R(680)+R(430)), R(860)/(R(550)×R(708)), (R(695-705))(-1)-(R(750-800))(-1), and REP-LEM (a index based on red edge position and estimated with a linear extrapolation method) were identified as optimum indices. Red-edge waveband (680-780 nm) appeared in all these optimum indices, indicating the importance of REP (red edge position) in chlorophyll estimation. When (R(695-705))(-1)-(R(750-800))(-1), the best index was applied to an independent validation set, chlorophyll content (r=0.8286) were reasonably well predicted, indicating model robustness. Depending on the sample, this technique enables to identify and characterize the relative content of various chlorophyll that distribution in the cucumber leaves. The map shows a relatively low level of chlorophyll at margins. Higher level can be noticed in the regions along the main veins and in some areas exhibiting dark green tissue. Our results indicate that hyperspectral imaging has considerable promise for predicting pigments in leaves and, the pigments can be detected in situ in living plant samples non-destructively.