Biotechnology and bioengineering of pullulanase: state of the art and perspectives.

Pullulanase (EC 3.2.1.41) is a starch-debranching enzyme in the α-amylase family and specifically cleaves α-1,6-glycosidic linkages in starch-type polysaccharides, such as pullulan, ß-limited dextrin, glycogen, and amylopectin. It plays a key role in debranching and hydrolyzing starch completely, thus bring improved product quality, increased productivity, and reduced production cost in producing resistant starch, sugar syrup, and beer. Plenty of researches have been made with respects to the discovery of either thermophilic or mesophilic pullulanases, however, few examples meet the demand of industrial application. This review presents the progress made in the recent years from the first aspect of characteristics of pullulanases. The heterologous expression of pullulanases in different microbial hosts and the methods used to improve the expression effectiveness and the regulation of enzyme production are also described. Then, the function evolution of pullulanases from a protein engineering view is discussed. In addition, the immobilization strategy using novel materials is introduced to improve the recyclability of pullulanases. At the same time, we indicate the trends in the future research to facilitate the industrial application of pullulanases.

Synthesis and Antibacterial Activity of Novel 4″-O-desosaminyl clarithromycin derivatives with 11, 12-arylalkyl side chains.

A series of novel 4″-O-desosaminyl clarithromycin derivatives with 11, 12-arylalkyl side chains was synthesized by coupling 6-deoxy-desosamine donors (18, 19) with 4″-OH of compounds 5a-c. The activities of the target compounds were tested against a series of macrolide-sensitive and macrolide-resistant pathogens. Some of them showed activities against macrolide sensitive and resistant pathogens, and compounds 21d and 21e displayed significant improvement of activities against resistant pathogens.

Antibacterial activities of a series of novel 5-O-(4', 6'-O-dimodified)-mycaminose 14-membered ketolides.

A series of novel 5-O-(4',6'-O-dimodified)-mycaminose 14-membered ketolides were assessed for their in vitro antibacterial activities against a panel of sensitive and resistant pathogens. Compound 1 and compound 2, two ester analogs, showed the best antibacterial activities against several macrolide-sensitive and macrolide-resistant strains. These results indicated that introducing ester to 6-OH and a small volume ether substituent to the 4-OH of mycaminose could improve the antibacterial activities of ketolides.

Facile synthesis of lipase-loaded starch nanoparticles as recyclable biocatalyst in Pickering interfacial systems.

To develop recyclable biocatalyst used in Pickering interfacial systems, the pH-responsive monomer [2-(dimethylamine)ethyl methacrylate] (DMAEMA) was grafted onto the maize starch molecule via free radical polymerization. Subsequently, combined with the gelatinization-ethanol precipitation and lipase (Candida rugosa) absorption process, an enzyme-loaded starch nanoparticle with DMAEMA grafting (D-SNP@CRL) was tailor-made, showing a nanometer size and regular sphere. X-ray photoelectron spectroscopy and confocal laser scanning microscopy confirmed a concentration-induced enzyme distribution within D-SNP@CRL, thereof the outside-to-inside enzyme distribution was proved to be optimum in achieving the highest catalytic efficiency. Benefited from the tunable wettability and size of D-SNP@CRL under pH variation, the generated Pickering emulsion could be readily applied as the recyclable microreactors for the n-butanol/vinyl acetate transesterification. This catalysis exhibited both highly catalytic activity and good recyclability, making the enzyme-loaded starch particle a promising green and sustainable biocatalyst in the Pickering interfacial system.

Berberine is a potential alternative for metformin with good regulatory effect on lipids in treating metabolic diseases.

Metformin (MTF) and berberine (BBR) share several therapeutic benefits in treating metabolic-related disorders. However, as the two agents have very different chemical structure and bioavailability in oral route, the goal of this study is to learn their characteristics in treating metabolic disorders. The therapeutic efficacy of BBR and MTF was systemically investigated in the high fat diet feeding hamsters and/or ApoE(-/-) mice; in parallel, gut microbiota related mechanisms were studied for both agents. We discovered that, although both two drugs had almost identical effects on reducing fatty liver, inflammation and atherosclerosis, BBR appeared to be superior over MTF in alleviating hyperlipidemia and obesity, but MTF was more effective than BBR for the control of blood glucose. Association analysis revealed that the modulation of intestinal microenvironment played a crucial role in the pharmacodynamics of both drugs, in which their respective superiority on the regulation of gut microbiota composition and intestinal bile acids might contribute to their own merits on lowering glucose or lipids. This study shows that BBR may be a good alternative for MTF in treating diabetic patients, especially for those complicated with dyslipidemia and obesity.

Synthesis, crystal structure and fluorescence of an unusual terbium carboxylate.

A novel terbium-containing fluorescent material, [Tb(CH3COO)2(H2O)3]Cl (1), has been prepared by a facile approach, i.e. an ultrasonic synthesis. Compound 1 has been structurally characterized via single-crystal X-ray diffraction and it crystallizes in the space group P21/n of the monoclinic system with four formula units in a cell. Compound 1 features a novel one-dimensional (1-D) chain-like structure. The 1-D chains are interconnected by hydrogen bonds to yield a 3-D supramolecular framework. Photoluminescent investigation reveals that compound 1 displays strong emission bands identified as the characteristic emissions of 5D4 → 7FJ (J = 3, 4, 5, 6) of Tb3+.

Impact of calcium ions and degree of oxidation on the structural, physicochemical, and in-vitro release properties of resveratrol-loaded oxidized gellan gum hydrogel beads.

Oxidized gellan gum (OGG) hydrogel beads as delivery systems for resveratrol were fabricated by ionic cross-linking with calcium chloride (CaCl2). The degree of oxidation (DO) and CaCl2 concentration had significant influences on the formation and functional properties of hydrogel beads. The resveratrol encapsulation efficiency (66.43%-79.84%) and loading capacity (4.15%-5.05%) of OGG hydrogel beads were enhanced as DO increased. The hydrogel beads exhibited a uniform spherical shape as observed by scanning electron microscope. Fourier transform infrared spectroscopy analysis confirmed that hydrogen bonds and ionic interaction participated in the formation of hydrogel beads. X-ray diffraction analysis revealed that the physical state of resveratrol was changed from crystalline to amorphous form after encapsulation. Furthermore, the physical stability and antioxidant capacity evaluation demonstrated that the hydrogel bead fabricated with DO80 OGG and CaCl2 concentration of 1.0 M could provide high protection for resveratrol against degradation by environmental stresses and maintain its antioxidant capacity. The DO and CaCl2 concentrations could modulate the in-vitro release behaviors of hydrogel beads and obtain a good small intestinal-targeted release of resveratrol at high DO and medium CaCl2 concentration. These findings suggested that a promising delivery system for encapsulating bioactive ingredients can be fabricated by rational design.

Preparation, physicochemical characterization and in vitro release behavior of resveratrol-loaded oxidized gellan gum/resistant starch hydrogel beads.

Hydrogel beads composed of oxidized gellan gum (OGG) and resistant starch (RS) were successfully fabricated by ionic cross-linking and used as delivery carriers for resveratrol. Firstly, OGG with different degrees of oxidation were prepared through 2, 2, 6, 6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation, and characterized by Fourier transform infrared spectroscopy and carbon-13 nuclear magnetic resonance to prove that carboxyl groups were successfully introduced into the gellan gum molecules. Molecular weight, thermal stability, zeta potential and gelation temperature of OGG were also investigated. Subsequently, resveratrol was encapsulated into OGG/RS hydrogel beads in the form of resveratrol/ß-cyclodextrins inclusion complexes. The addition of RS significantly influenced the morphological structure and swelling capacity of OGG/RS hydrogel beads. The OGG/RS hydrogel beads exhibited a pH-sensitivity and high encapsulation efficiency of resveratrol (84.95 %-90.73 %). Furthermore, the in-vitro release behaviors demonstrated that OGG/RS hydrogel beads showed good stability in simulated gastric fluids and sustained release of resveratrol in simulated intestinal fluids. The obtained results indicate that OGG/RS hydrogel beads show a potential as delivery system for resveratrol in the food industry.

Spiral-Dextrin Complex Crystals: Efficient Approach for Colon-Targeted Resveratrol Delivery.

In this work, spiral dextrin/resveratrol (SD/Res) crystal, a new colon-specific drug-delivery system, was established by a novel method of encapsulation and cocrystallization to improve the antidigestion ability compared with the SD/Res inclusion complex (SD/Res IC) prepared by encapsulation and coprecipitation. X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed that the SD/Res crystal formed a more regular and perfect crystallite than SD/Res IC. Moreover, the encapsulation ability and thermostability of the SD/Res crystal were enhanced as the chain length of SD was increased. In vitro digestion indicated that SD/Res IC merely achieved small intestine-targeted release of resveratrol, while the SD/Res crystal could act as a colon-specific delivery system to protect resveratrol from degradation by gastric acid and pancreatic enzymes. The SD-1/Res crystal presented much higher thermal stability and stronger gastrointestinal stability than other SD/Res crystals and SD/Res ICs, which facilitated its application as a novel colon-target delivery system for resveratrol.

Structural characterization, anticancer, hypoglycemia and immune activities of polysaccharides from Russula virescens.

Russula virescens is an edible wild mushroom that is widely distributed in south of China. This research aimed to analyze the structure characterization and evaluate the hypoglycemic, anticancer and immunological activities of two water soluble polysaccharides RVP-1 and RVP-2 from R. virescens. The results showed RVP-1 and RVP-2 were non-triple helix structured hetero-polysaccharides with different weight-average molecular weight 14,883 and 13,301 Da, respectively. Both RVP-1 and RVP-2 were composed of galactose, glucose, mannose and fructose, and the sugar residues were mainly linked by 1,6→, 1,2→, 1→ and 1,3,6→ glycosidic bonds. Moreover, the antidiabetic, anticancer and immune activities of RVP-1 and RVP-2 were explored in vitro methods. The two polysaccharides have potential for inhibiting α-glucosidase and α-amylase activities, suppressing HepG-2, A549 and MCF-7 cancer cells proliferation, and activating macrophage RAW 264.7 cells to secret immune cytokines for mediating cellular immune response. These findings provided a scientific basis for further utilization of polysaccharide from R. virescens.

X(3915) and X(4350) as new members in the P-wave charmonium family.

The analysis of the mass spectrum and the calculation of the strong decay of P-wave charmonium states strongly purport to explain the newly observed X(3915) and X(4350) as new members in the P-wave charmonium family, i.e., chi{c0}{'} for X(3915) and chi{c2}{''} for X(4350). Under the P-wave charmonium assignment to X(3915) and X(4350), the J{PC} quantum numbers of X(3915) and X(4350) must be 0{++} and 2{++} respectively, which provide important criteria to test the P-wave charmonium explanation for X(3915) and X(4350) proposed by this Letter. The decay behavior of the remaining two P-wave charmonium states with the second radial excitation is predicted, and an experimental search for them is suggested.

Effects of octenyl succinic anhydride groups distribution on the storage and shear stability of Pickering emulsions formulated by modified rice starch.

The modified rice starches by octenyl succinic anhydride (OS-RSs) with the same degree of substitution of 0.033 were prepared in aqueous solution using 3% or 20% (w/w) NaOH as a catalyst (OS-RS 3 or OS-RS 20). The results indicated that 20% of NaOH could destroy the structure of starch more seriously and led to more OS groups travelling to interior of starch granules. Furthermore, the storage stability and rheological properties of Pickering emulsions formulated by RS, OS-RS 3 and OS-RS 20 were investigated. Emulsion index of OS-RS 20 were always lower than OS-RS 3 during the storage time, while G', G" and viscosity of O/W Pickering emulsion formulated by OS-RS 20 particles were greater than those of OS-RS 3. OS-RS 3 with more hydrophobic groups in the granule surface could form more compact oil/water layers which contributed greater to the storage stability of Pickering emulsion due to the formation of more compact oil/water layers. However, OS-RS 20 with homogeneous distribution of OS groups in the whole particles resulted in the formation of a tighter droplet network structure that was more resistant to external impacts, which showed OS-RS 20 could improve the shear stability of Pickering emulsion.

Co-encapsulation of Vitamin C and ß-Carotene in liposomes: Storage stability, antioxidant activity, and in vitro gastrointestinal digestion.

Vitamin C (VC) and ß-Carotene (ßC) were selected to produce co-encapsulated liposomes using hydrophilic and hydrophobic cavities simultaneously by ethanol injection method. The results of liposomal structure characterized by particle size, polydispersity index, zeta-potential and transmission electron microscope showed that the microstructure of all liposomal samples was spherical without adhesion or break and the size of VC-ßC-loaded liposome (L-VC-ßC) was bigger than VC-loaded liposome (L-VC) or ßC-loaded liposome (L-ßC). The encapsulation efficiency (EE) of VC in L-VC-ßC was significantly higher than that in L-VC, and the EE of ßC in L-VC-ßC had no significant change compared with that in L-ßC. The free radical scavenging rate of L-VC-ßC was significantly higher than that of L-ßC, while it had no significant change compared with that of L-VC. In addition, the storage stability of ßC in L-VC-ßC improved greatly compared with that in L-ßC. Furthermore, the zero order model was applied to understand the release kinetics of ßC from L-ßC and L-VC-ßC in the stomach, whereas the Korsmeyr-Peppas model was chosen to describe the release of ßC from two types of liposome in small intestine and their release mechanisms were mainly dominated by Fickian diffusion. It was significant to provide a new idea for using hydrophilic and hydrophobic cavities simultaneously in liposomes to design the multicomponent nutrient delivery system.

[The optimization and assessment of the method for inducing hyperuricemia in rats].

Objective: To explore an effective method for inducing a rat model with hyperuricemia in a short period and assess the effects of the model. Methods: Sprague-Dawley rats were adopted as donors and randomly divided into control group (CT group, n=6) and 5 model groups (M1-M5 groups, n=8 in each group). M1 group (gavage with 10 g/kg yeast extracts and 100 mg/kg adenine, twice per day, 300 mg/kg oxonic acid potassium by intraperitoneal injection, in the 7th day of model inducing), M2 group (gavage with 10 g/kg yeast extracts and 100 mg/kg adenine, twice per day, 300 mg/kg oxonic acid potassium by intraperitoneal injection, in the 1st, 3rd and 7th day of model inducing),M3 group (gavage with 10 g/kg yeast extracts and 100 mg/kg adenine, twice per day, 300 mg/kg oxonic acid potassium by intraperitoneal injection, once per day during the model inducing), M4 group (gavage with 20 g/kg yeast extracts and 100 mg/kg adenine, twice per day, 300 mg/kg oxonic acid potassium by intraperitoneal injection, once per day during the model inducing), M5 group (gavage with 30 g/kg yeast extracts and 100 mg/kg adenine, twice per day, 300 mg/kg oxonic acid potassium by intraperitoneal injection, once per day during the model inducing), and group CT (gavaged with equal volume sterilized water and intraperitoneal injected with normal saline according to the weight and at the same frequency as the model groups). The model inducing lasted for 7 days. After the inducing was finished, blood and 24-hour urine were sampled for uric acid and creatinine determination. Then rats were maintained for 2 weeks and blood and 24-hour urine samples were collected, the concentration of uric acid and creatinine were detected. The kidney and stomach were weighed,morphological changes in kidney were observed. Results: After model inducing, the body weight of rats in all model groups was lower than that of the control group (P＜0.01). Deaths occurred in all the rats with model treatments except M2. M4 and M5 groups were failed to be analyzed because of the high mortality, model 1 and 3 groups had 4 and 2 deaths, respectively. The uric acid levels in blood and urine of the model groups were significantly elevated (P＜ 0.01) at the end of model inducing. The model 2 group's blood uric acid was highest among the model groups (P＜0.05). It sustained a higher concentration than CT group in the three model groups after 2 weeks feeding (P＜0.05). The kidneys in model groups obviously swelling and were heavier than CT group (P＜0.01). The inflammation and structural damages were observed in kidneys of all model groups.Conclusion: The yeast extract (10 g/kg), adenine (100 mg/kg) gavage combined with intraperitoneal injections(the 1st, 3rd, 7th day during inducing) of potassium oxonate can be an rapid and effective method for inducing the rat model with hyperuricemia, which can be suggested to the related research.

Total synthesis of (-)-talaumidin and (-)-galbelgin.

( - )-Talaumidin (1) and ( - )-galbelgin (2) have been synthesized via 4-pentenoic acid as a starting material with the overall yield of about 17.8 and 16.9%, respectively. The key steps include Evans asymmetry anti-aldol reaction, TBS protection, hydroboration, oxidation, Friedel-Crafts arylation, etc.

Clinical value of whole-body magnetic resonance diffusion weighted imaging on detection of malignant metastases.

OBJECTIVE: To evaluate the value of whole-body diffusion weighted imaging (WB-DWI) on detection of malignant metastasis. METHODS: Forty-six patients with malignant tumors underwent WB-DWI examinations between April 2007 and August 2007 in our hospital. Before WB-DWI examination, the primary cancers of all the patients were confirmed by pathology, and the TNM-stage was assessed with conventional magnetic resonance imaging (MRI) or computed tomography (CT). WB-DWI was performed using short TI inversion recovery echo-planar imaging (STIR-EPI) sequence. Abnormal high signal intensities on WB-DWI were considered as metastases. The results of WB-DWI were compared with other imaging modalities. For the assessment of the diagnostic capability of WB-DWI, WB-DWI were compared with CT for demonstrating mediastinal lymph node metastases and lung metastases, and with conventional MRI for demonstrating metastases in other locations. RESULTS: WB-DWI demonstrated 143 focuses, 14 of which were diagnosed to be benign lesions in routine imaging. The number of bone metastases depicted on WB-DWI and routine imaging was 85 and 86; lymph node metastases was 17 and 18; liver metastases was 14 and 14; lung metastases was 4 and 8; and brain metastases was 6 and 8, respectively. WB-DWI failed to detect 12 metastatic lesions including 3 osteoplastic bone metastases, 4 lung metastases, 3 mediastinal lymph node metastases, and 2 brain metastases. Four metastatic lesions including 2 deltopectoral lymph nodes and 2 rib metastases were detected with WB-DWI alone, all of which evolved greatly during clinical follow-up for more than 6 months. WB-DWI had higher detection rates for metastatic lesions in liver, bone, and lymph nodes than those in lung and brain (chi2=30, P<0.001). CONCLUSIONS: WB-DWI could detect most of metastatic lesions that were diagnosed with conventional MRI and CT. The limitations of WB-DWI might be had high false-positive rate and low efficiency in detecting mediastinal lymph node, brain, and lung metastases.

Fine structure, crystalline and physicochemical properties of waxy corn starch treated by ultrasound irradiation.

As a simple and effective physical method, ultrasound irradiation has been used to modify starch. Native waxy corn starch was treated by ultrasound irradiation at 100 and 400 W in this study. Compared with native waxy corn starch, lower proportion of B1, B2, and B3, higher proportion of A chain were observed in ultrasonicated waxy corn starch. 1H NMR combined with HPSEC-MALLS-RI data showed that lower degree of branching was observed in ultrasonicated waxy corn starch, and α-1,4 glycosidic linkages were more stable than α-1,6 glycosidic linkages in waxy corn starches. 13C NMR data indicated that the content of double helices was decreased, and single helix and amorphous components were increased after ultrasound irradiation. The A-type crystal structure was scarcely affected according to X-ray diffraction (XRD) analysis. The granule surface of ultrasonicated waxy corn starch became notch and rough fragment, and lower particle diameter was observed in ultrasonicated waxy corn starch. These results demonstrated that ultrasound irradiation affected chain length distribution, double helices, single helices and amorphous state, especially α-1,4 glycosidic linkages and α-1,6 glycosidic linkages, of waxy corn starch.

A comparison study on polysaccharides extracted from Fructus Mori using different methods: structural characterization and glucose entrapment.

In this study, the structure characteristics and the hypoglycemic and antioxidant activities of mulberry fruit polysaccharides obtained by the commonly used hot water (MFPh)-, ultrasonic (MFPu)-, acid (MFPc)- and alkali (MFPa)-assisted extraction methods were investigated. NMR analysis indicated that the four polysaccharides had similar glycosidic linkage patterns. Scanning electron microscopy analyses showed that the surface morphology of the polysaccharides was greatly affected by the extraction methods. The results of the bioactivity assays indicated that MFPh exhibited stronger antioxidant and α-amylase inhibitory activities than the other polysaccharides. Moreover, all the polysaccharides showed good α-glucosidase inhibitory activities except for MFPu with the lowest molecular weight. These results suggested that acid, alkali, and ultrasonic-assisted extractions have different effects on the degradation of polysaccharides without changing the main structure compared with hot water extraction. In addition, the molecular weight of polysaccharides plays a key role in the bioactivity of the mulberry fruit polysaccharides.

Liver-target nanotechnology facilitates berberine to ameliorate cardio-metabolic diseases.

Cardiovascular and metabolic disease (CMD) remains a main cause of premature death worldwide. Berberine (BBR), a lipid-lowering botanic compound with diversified potency against metabolic disorders, is a promising candidate for ameliorating CMD. The liver is the target of BBR so that liver-site accumulation could be important for fulfilling its therapeutic effect. In this study a rational designed micelle (CTA-Mic) consisting of α-tocopheryl hydrophobic core and on-site detachable polyethylene glycol-thiol shell is developed for effective liver deposition of BBR. The bio-distribution analysis proves that the accumulation of BBR in liver is increased by 248.8% assisted by micelles. Up-regulation of a range of energy-related genes is detectable in the HepG2 cells and in vivo. In the high fat diet-fed mice, BBR-CTA-Mic intervention remarkably improves metabolic profiles and reduces the formation of aortic arch plaque. Our results provide proof-of-concept for a liver-targeting strategy to ameliorate CMD using natural medicines facilitated by Nano-technology.

[Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells].

OBJECTIVE: To study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells. METHODS: Three pairs of 21 bp reverse repeated motifs of CENP-A target sequence with 9 spacer were synthesized and inserted into vector pSilencer 2.1-U6 neo to generate siRNA eukaryotic expression plasmids. After stable transfection into HepG2 cells, cell growth, apoptosis, cell cycles and plate clone forming efficiency were investigated. Expressions of CENP-A mRNA was monitored by the reverse transcriptase polymerase chain reaction (RT-PCR). The protein expression of CENP-A, bcl-2, Bax, p53, p21waf1 and mdm2 were detected by Western-blotting. RESULTS: Two eukaryotic expression plasmids with significant siRNA specific inhibition to the CENP-A gene were created. Compared with control cells, HepG2 cells transfected with the constructs showed G1 phase delay (P < 0.01) and cell number decrease in the S phase (P < 0.001), along with an increased apoptotic rate (P = 0.003), significant increase of Bax expression and decreased bcl-2 expression (P< or =0.001). The protein expressions of p21waf1 was higher and mdm2 was lower than those of the control groups. However, the wild type p53 protein expression was not effected by CENP-A siRNA. CONCLUSIONS: An altered expression of CENP-A may be related to the proliferation of hepatocellular carcinoma through cell cycle regulation involving an altered bcl-2/Bax expression, that may be p53 independent.