Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

Tamarind Xyloglucan Oligosaccharides Attenuate Metabolic Disorders via the Gut-Liver Axis in Mice with High-Fat-Diet-Induced Obesity.

Functional oligosaccharides exert obesity-reducing effects by acting at various pathological sites responsible for the development of obesity. In this study, tamarind xyloglucan oligosaccharides (TXOS) were used to attenuate metabolic disorders via the gut-liver axis in mice with high-fat-diet (HFD)-induced obesity, as determined through LC/MS-MS and 16S rRNA sequencing technology. A TXOS dose equivalent to 0.39 g/kg/day in humans restored the gut microbiota in obese mice, which was in part supported by the key microflora, particularly Bifidobacterium pseudolongum. Moreover, TXOS reduced the abundance of opportunistic pathogen species, such as Klebsiella variicola and Romboutsia ilealis. The bodyweight and weight gain of TXOS-treated (4.8 g/kg per day) mice began to decrease at the 14th week, decreasing by 12.8% and 23.3%, respectively. Sixteen fatty acids were identified as potential biomarkers in the liver, and B. pseudolongum and caprylic acid were found to tightly regulate each other. This was associated with reduced inflammation in the liver, circulation, and adipose tissue and protection from metabolic disorders. The findings of this study indicate that TXOS can significantly increase the gut microbiota diversity of obese mice and restore the HFD-induced dysbiosis of gut microbiota.

One-Pot Three-Enzyme System for Production of a Novel Prebiotic Mannosyl-ß-(1 → 4)-Fructose Using a d-Mannose Isomerase from Xanthomonas phaseoli.

The present supply of prebiotics is entirely inadequate to meet their demand. To produce novel prebiotics, a d-mannose isomerase (XpMIaseA) from Xanthomonas phaseoli was first produced in Komagataella phaffii (Pichia pastoris). XpMIaseA shared the highest amino acid sequence identity (58.0%) with the enzyme from Marinomonas mediterranea. Efficient secretory production of XpMIaseA (282.0 U mL-1) was achieved using high cell density fermentation. The optimal conditions of XpMIaseA were pH 7.5 and 55 °C. It showed a broad substrate specificity, which isomerized d-mannose, d-talose, mannobiose, epilactose, and mannotriose. XpMIaseA was employed to construct a one-pot three-enzyme system for the production of mannosyl-ß-(1 → 4)-fructose (MF) using mannan (5%, w/v) as the substrate. The equilibrium yield of MF was 58.2%. In in vitro fermentations, MF significantly stimulated (≤3.2-fold) the growth of 12 among 15 tested Bifidobacterium and Lactobacillus strains compared with fructo-oligosaccharides. Thus, the novel d-mannose isomerase provides a one-pot bioconversion strategy for efficiently producing novel prebiotics.

A novel neutral thermophilic ß-mannanase from Malbranchea cinnamomea for controllable production of partially hydrolyzed konjac powder.

Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel ß-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 ß-mannanases. Through high cell density fermentation, the highest ß-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75 °C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of ß2-α2 loop, McMan5B showed unique hydrolysis property as compared to other ß-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000 Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable ß-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: • A novel ß-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. • McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. • PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.

Protective effects of lotus plumule ethanol extracts on bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a progressive fibrosing disease, characterized by excessive accumulation of extracellular matrix (ECM) and remodeling of the lung architecture, which finally result in respiratory failure. Currently, there is no satisfactory treatment for PF， therefore, the development of effective agents is urgently needed. Lotus plumule, the green embryo of Nelumbo nucifera Gaertn., a plant of the Nymphaeaceae family, is a traditional Chinese food with exceptional nutritional value and its extracts exert prominent anti-inflammatory and anti-fibrotic effects. The aim of the present study was to investigate the inhibitory effects of lotus plumule extracts (LPEs) on bleomycin (BLM)-induced PF in mice. Therefore, enzyme-linked immunosorbent assay, RT-PCR, and western blot analysis were performed. The histopathological examination demonstrated that LPEs could obviously decrease the degree of alveolitis, deposition of ECM and the production of collagen I (Col-I) in the pulmonary interstitium. In addition, the results showed that LPEs markedly alleviated the expression of interleukin (IL)-6, IL-17, transforming growth factor (TGF)-ß, and α-smooth muscle actin (α-SMA). Additionally, the content of Col-I and hydroxyproline (HYP) was also attenuated. In conclusion, LPEs could ameliorate the BLM-induced lung fibrosis, thus suggesting that LPEs could serve as a potential therapeutic approach for PF.

High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.

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.

Preparation, characterization, and prebiotic activity of manno-oligosaccharides produced from cassia gum by a glycoside hydrolase family 134 ß-mannanase.

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 ß-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-ß-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.

MicroRNA­145 inhibits migration and induces apoptosis in human non­small cell lung cancer cells through regulation of the EGFR/PI3K/AKT signaling pathway.

In the present study, the therapeutic effects and the underlying molecular mechanisms of microRNA (miR)­145 were investigated in non­small cell lung cancer (NSCLC) cells. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to examine miR­145 expression. An MTT assay and flow cytometry were used to investigate cell proliferation and apoptosis, respectively. The protein expression of Bax, epidermal growth factor receptor (EGFR), phosphatidylinositol 3­kinase (PI3K) and phosphorylated­protein kinase B (AKT) was examined by western blot analysis. miR­145 expression was downregulated in patients with NSCLC who were treated with chemotherapy. The downregulation of miR­145 in A549 cells reduced lactate dehydrogenase (LDH) expression, apoptosis, caspase­3/-9 levels and Bax protein expression, while it increased cell proliferation. Upregulation of miR­145 in A459 cells increased LDH, apoptosis, caspase­3/-9 levels and Bax protein expression, while it inhibited cell proliferation. The EGFR/PI3K/AKT signaling pathway was suppressed by miR­145 upregulation in A549 cells and induced by miR­145 downregulation. The EGFR inhibitor suppressed the EGFR/PI3K/AKT signaling pathway and increased the anticancer effects of miR­145 upregulation in A549 cells. The PI3K inhibitor suppressed the PI3K/AKT signaling pathway and reversed the anticancer effects of miR­145 upregulation in A549 cells. In conclusion, the present study demonstrated that miR­145 regulates the EGFR/PI3K/AKT signaling pathway in patients with NSCLC.

Structural and biochemical insights into the substrate-binding mechanism of a novel glycoside hydrolase family 134 ß-mannanase.

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. ß-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-ß-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 ß-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.

High-level expression of an engineered ß-mannanase (mRmMan5A) in Pichia pastoris for manno-oligosaccharide production using steam explosion pretreated palm kernel cake.

An engineered ß-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 ß-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.

Over-regulation of microRNA-133b inhibits cell proliferation of cisplatin-induced non-small cell lung cancer cells through PI3K/Akt and JAK2/STAT3 signaling pathway by targeting EGFR.

The present study determined the anticancer activity and its mechanism of microRNA­133b on cell proliferation of cisplatin-induced non-small cell lung cancer cells. The expression of microRNA-133b cisplatin­induced non-small cell lung cancer (NSCLC) tissue was lower than that of para-carcinoma tissue in patients. Overall survival of higher expression in cisplatin-induced NSCLC patients was higher than that of lower expression in cisplatin­induced NSCLC patients. Over-regulation of microRNA-133b inhibited cell proliferation and LDH activity, induced apoptosis and caspase-3 activity, suppressed the protein expression of EGFR, PI3K, p-Akt, p-JAK2 and p-STAT3, decreased cyclin D1 and increased Bax protein expression in cisplatin­induced A549 cells. EGFR inhibitor (lapatinib) suppressed EGFR protein expression, inhibited cell proliferation and LDH activity, and induced apoptosis and caspase-3 activity in cisplatin-induced A549 cells by over-regulation of microRNA-133b. When EGFR protein expression was suppressed, PI3K, p-Akt, p-JAK2 and p-STAT3, decreased cyclin D1 and increased Bax protein expression in cisplatin-induced A549 cells by over-regulation of microRNA-133b. Altogether, our results indicated that over-regulation of microRNA-133b inhibits cell proliferation of cisplatin-induced NSCLC by PI3K/Akt and JAK2/STAT3 signaling pathway by targeting EGFR.

High level expression of ß-mannanase (RmMan5A) in Pichia pastoris for partially hydrolyzed guar gum production.

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel ß-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<7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6×104Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-d-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable ß-mannanase for PHGG production and some useful information of PHGG are provided in this paper.

Directed evolution of a ß-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions.

BACKGROUND: ß-Mannanase randomly cleaves the ß-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of ß-mannanase have tremendously expanded in recent years, the wild-type ß-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 ß-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 ß-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 (kcat/Km) 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 ß-mannanase was acquired, which could be used for modifying ß-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a ß-mannanase from zygomycete engineered by directed evolution.

[Study on Regulation Effects of Yiqiyangyin Formula on Advanced Lung Cancer Cachexia Mice].

Objective: To observe the effect of Yiqiyangyin formula on advanced lung cancer cachexia mice. Methods: 60 C57 BL /6inbred mice( SPF) were randomly divided into normal control group, model group, Yiqiyangyin formula group,indomethacin group, and cisplatin group, with 12 mice in each group. Model group and normal control group were treated with the same amount of normal saline. Except for the normal control group, the mice of the other groups were built with Lewis lung cancer model. The changes of the level of body mass, feed consumption,serum cytokine,the expression of TNF-α and its receptor were observed. Results: After eight days of treatment, compared with cisplatin group, the levels of CEA,NSE,Pro GRP in serum,CD3+,CD4+,CD4+/ CD8+levels in serum peripheral blood and TNF-R1 expressions in tumor tissues of Yiqiyangyin formula group were increased( P < 0. 05),the contents of TNF-α and IL-6 and the expressions of TNF-α and TNF-R2 in tumor tissues of Yiqiyangyin formula group were significantly decreased( P <0. 05). Conclusion: Yiqiyangyin formula affects on advanced lung cancer cachexia, which may be related to the regulation of the immune and inflammatory cytokines expression.

[Localization of the bronchial air leakage in pneumothorax by injection of human albumin foam].

OBJECTIVE: To evaluate the efficacy and safety of a new method to determine the bronchus for air leakage in pneumothorax by injection of human albumin foam. METHODS: In 29 cases with pneumothorax, the bronchus responsible for air leakage was localized by injecting foam of human albumin into target bronchus under direct view of bronchoscopy. RESULTS: The bronchus for air leakage was successfully localized in all the 29 cases of pneumothorax. The average time for locating (from injection of the foam to the localization of the bronchus) was (4.0 ± 1.2) min, and the average amount of 20% human albumin used was (8.0 ± 2.6) ml for each patient. The air leakage was treated accordingly, and occlusion by fibrin glue was successfully carried out in 21 cases and by OB glue in 8 cases. Severe cough was noted in 6, fever in 4, thoracic bleeding in 4 cases, and chest pain in 1 case. CONCLUSION: Injection of human albumin foam into target bronchus under bronchoscopy was a simple, safe and effective method for the localization of the bronchus for air leakage in pneumothorax.

Characterization of p70 S6 kinase 1 in early development of mouse embryos.

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one-cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M-phase promoting factor (MPF) and delayed the first mitotic cleavage. Down-regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one-cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho-S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos.

[Near infrared determination of sugar content in apples based on GA-iPLS].

To improve and simplify the prediction model of sugar content, genetic algorithm interval partial least square (GA-iPLS) methods, the evolution of iPLS described by Lars Nørgaard, were proposed and used to establish the calibration models of sugar content against apple spectra. The apple spectra data were divided into 40 intervals, among which 5 subsets, i. e. No. 4, 6, 8, 11 and 18, containing 362 data points were selected by GA-iPLS. The optimum GA-iPLS calibration model was obtained with the correlation coefficient (rc) of 0.962, the root mean square error of cross-validation (RMSECV) of 0.3346 and the root mean square error of prediction (RMSEP) of 0.3846. Compared with the whole spectra data model, the data points and the factors in the GA-iPLS were decreased significantly. Consequently, the running time of the PLS model build by GA-iPLS was shorter than that of the whole spectra data model. Furthermore, the GA-iPLS model could not only improve precision, but also simplify the model.

Expression and immunohistochemical localization of Cdc2 and P70S6K in different stages of mouse germ cells.

In order to determine the function and possible relationship between Cdc2 and P(70)S6K, Western blot analysis and immunohistochemistry analysis were used to study the expression and kinase activity of Cdc2 and P(70)S6K in male mouse germ cells. With the maturation of germ cells in the testis, the expression of Cdc2 and P(70)S6K was relatively constant. However, the kinase activity of P(70)S6K was increased and the phosphorylation of Tyr15 residue of Cdc2 was enhanced, which suggests that the kinase activity of Cdc2 is decreasing. Immunohistochemistry analysis also showed that there was a P(70)S6K transfer from nucleus to cytoplasm during spermatogenesis. During spermatogenesis, cell division of the germ cell in male mouse is decelerated; nevertheless, cell growth is enhanced. Cdc2 and P(70)S6K are involved in these two processes. It could be an alternative mechanism to prepare for future fertilization that Cdc2 is able to maintain a subtle balance between the production and growth of male germ cells by regulating P(70)S6K.