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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.

The digestibility and prebiotic potential of galactomannan oligosaccharides prepared from Cassia seed gum (CMOS) was studied. CMOS were tolerant against simulated gastrointestinal digestion. During in vitro human fecal fermentation with CMOS, the microbiota composition was significantly changed with promoted growth of potential beneficial genera (i.e. Bifidobacterium, Lactobacillus, Bacteroides, Veillonella) while that of potential harmful genera (i.e. Fusobacterium, Lachnospiraceae, and Sutterella) was inhibited. Short chain fatty acids were produced with decreased medium pH, while acetic acid (20.85 mM) and propionic acid (19.77 mM) were the predominant metabolites. For CMOS utilization, α-galactosidase and β-mannosidase were involved with the enzyme activity reached the maximum after 3 h fermentation. The HPAEC chromatograms showed that CMOS with DP = 3 and 4 were prior utilized through enzymatic hydrolysis within 36 h fermentation. Findings gained here will introduce the utilization and targeted production of CMOS (DP = 3 and 4) as prebiotics with defined structural features.

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.

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.

An engineered β-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680 U mL−1. The mRmMan5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper.

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.

Functional oligosaccharides, particularly konjac mannan oligosaccharides (KMOS), can regulate glucose metabolism. However, the molecular mechanisms involved in the hypoglycemic effect of KMOS remain largely unknown. Here, the effect of KMOS supplementation on glucose homeostasis was evaluated in both high-fat diet (HFD)-fed C57BL/6J mice and high-glucosamine-induced HepG2 cells. KMOS supplementation remarkably ameliorated the fasting blood glucose, glucose tolerance, and insulin tolerance of HFD-fed mice. Abnormalities of triglyceride and glycogen metabolism in the liver induced by the HFD were reversed by KMOS supplementation. The insulin signaling pathway was activated by KMOS, with stimulation of GLUT2 membrane translocation and glucose uptake in HepG2 cells via the AMPK pathway. Moreover, KMOS suppressed p-mTOR expression and stimulated the GSK-3&beta, /CREB pathway via the AMPK pathway. KMOS significantly upregulated leptin receptor expression and downregulated PTP1B and SOCS3 levels in the liver and brain, with a decreased serum leptin concentration. Phosphorylation of JAK2 and STAT3 in the liver was activated by KMOS supplementation, while the expressions of Sirt1, Tfam, and Pgc1-&alpha, in the brain were elevated. Conclusively, KMOS attenuated HFD-induced glucose metabolism dysfunction through the regulation of insulin resistance and leptin resistance. This finding indicates that KMOS have potential value as an anti-hyperglycemic dietary supplement.

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.

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.

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.