Search

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.

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.

β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme–manno-oligosaccharides from this family has been reported. Here, crystal structures of a GH family 113 β-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had higher affinity for −1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (−4 to +2) exist in the groove, but mannose units preferentially occupied subsites −4 to −1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH family 113 β-1,4-mannanase–manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH family 113 members, and provide novel insight into protein engineering of GHs to improve their hydrolysis activity.

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.

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.

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.