We report a novel “anchor–shield” approach for synthesizing a yolk–shell-structured biocatalytic system that consists of a phenylalanine ammonia lyase (PAL) protein particle core and a hollow silica shell with large mesopores by a combination of CaCO3 microtemplates and biomimetic silicification. The method is established upon filling porous CaCO3 cores with PAL via co-precipitation, controlled self-assembly and polycondensation of silanes, cross-link of the PAL molecules, and subsequent CaCO3 dissolution. During this process, the self-assembled layer of cetyltrimethylammonium bromide served as a structure-directing agent of the mesostructure and directed the overgrowth of the mesostructured silica on the external surface of PAL/CaCO3 hybrid microspheres; after CaCO3 dissolution, the cross-linked PAL particles were encapsulated in the hollow silica shell. The hollow silica shell around the enzyme particles provided a “shield” to protect from biological, thermal, and chemical degradation for the enzyme. As a result, the recycling of the PAL enzyme was improved remarkably in comparison to adsorbed PAL on CaCO3. PAL particles with a hollow silica shell still retained 60% of their original activity after 13 cycles, whereas adsorbed PAL on CaCO3 microparticles lost activity after 7 cycles. Moreover, immobilized PAL exhibited higher stability against a proteolytic agent, denaturants, heat, and extreme pH than adsorbed PAL on CaCO3 microparticles. These results demonstrated that the “anchor–shield” approach is an efficient method to obtain a stable and recycled biocatalyst with a yolk–shell structure.Keywords: biomimetic silicification; CaCO3 microtemplates; enzyme immobilization; hollow silica shell; stability;

Metal–organic frameworks (MOFs) have recently emerged as a promising candidates for the immobilization of enzymes due to their diversified structures and porosity. However, a lack of good size and morphological control over the as-prepared MOFs has limited their practical applications in some cases. Herein, instead of zeolitic imidazolate framework-8 (ZIF-8) with the standard rhombic dodecahedral morphology, we successfully synthesize a novel mesoporous catalase@ZIF composite with cruciate flower-like morphology by embedding catalase molecules into uniformly sized ZIF crystals. With extraordinarily large mesopore size and high protein loading capacity, the catalase@ZIF composites with cruciate flower-like morphology exhibit 400% higher activity than that of catalase@ZIF composites with conventional rhombic dodecahedral morphology, and show higher reusability than conventional rhombic dodecahedral morphology. More importantly, we demonstrate for the first time that the biomacromolecules (proteins) can not directly regulate the crystal size, morphology, and crystallinity of ZIF-8. Moreover, the crystal morphology of ZIF has primary dependence on concentrations of 2-methylimidazole and Zn2+ ions, and can be directly controlled by adjusting concentrations of Zn2+ ions while keeping the high concentration of 2-methylimidazole.Keywords: enzyme immobilization; metal−organic frameworks; morphological control; nanobiocatalysis; nanomaterials;

Metal–organic frameworks (MOFs) have recently emerged as a promising candidates for the immobilization of enzymes due to their diversified structures and porosity. However, a lack of good size and morphological control over the as-prepared MOFs has limited their practical applications in some cases. Herein, instead of zeolitic imidazolate framework-8 (ZIF-8) with the standard rhombic dodecahedral morphology, we successfully synthesize a novel mesoporous catalase@ZIF composite with cruciate flower-like morphology by embedding catalase molecules into uniformly sized ZIF crystals. With extraordinarily large mesopore size and high protein loading capacity, the catalase@ZIF composites with cruciate flower-like morphology exhibit 400% higher activity than that of catalase@ZIF composites with conventional rhombic dodecahedral morphology, and show higher reusability than conventional rhombic dodecahedral morphology. More importantly, we demonstrate for the first time that the biomacromolecules (proteins) can not directly regulate the crystal size, morphology, and crystallinity of ZIF-8. Moreover, the crystal morphology of ZIF has primary dependence on concentrations of 2-methylimidazole and Zn2+ ions, and can be directly controlled by adjusting concentrations of Zn2+ ions while keeping the high concentration of 2-methylimidazole.Keywords: enzyme immobilization; metal−organic frameworks; morphological control; nanobiocatalysis; nanomaterials;

Culture conditions significantly influence extracellular polysaccharide (EPS) production of Nostoc flagelliforme, however, the key enzyme controlling EPS synthesis has not been fully explored yet. The influence of different culture conditions including light quality, carbon source and nitrogen source on EPS production of N. flagelliforme and activities of EPS synthesis enzymes was investigated. Three experimental groups produced higher amounts of EPS than the control group, including the carbon source group with 1.26 g L−1 NaHCO3, the nitrogen source group with 0 g L−1 NaNO3 and the light quality group with blue light. Activities of seven related enzymes phosphoglucose isomerase (PGI), fructose-1,6-bisphosphatase (FBPase), UDP-glucose pyrophosphorylase (UGPase), UDP-galactose-4-epimerase (UGE), UDP-glucose dehydrogenase (UGDH), phosphomannose isomerase (PMI), and phosphofructokinase (PFK) were significantly influenced by culture conditions. Partial least-squares analysis and correlation analysis methods were used to analyze the relationship between the activities of these enzymes and EPS production, and a correlation between the production of EPS and the activities of PGI, PMI, FBPase, UGDH, and UGPase was found under different culture conditions. Subsequent analysis of the transcription level of genes encoding the five enzymes showed genes pgi and fbp1 in three experimental groups were significantly up regulated. The results revealed PGI and FBPase might be important enzymes positively influencing the biosynthesis of N. flagelliforme EPS. The findings would be helpful to further understand the pathway of EPS biosynthesis aimed to improve the EPS production of N. flagelliforme.

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising tool for enzyme immobilization because of their simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications. For example, the prepared CLEAs exhibit amorphous large clusters that would cause significant mass-transfer limitations, which lead to a low catalytic efficiency. Here, inspired by biomineralized core–shell structures in nature, we develop a novel mesoporous spherical CLEA with a biosilica shell by using phenylalanine ammonia lyase based on CaCO3 microtemplates and biomimetic mineralization. The resultant CLEAs exhibited a spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs and showed activity higher than that of conventional CLEAs. Moreover, the thermostability, tolerance against denaturants, and mechanical stability of the spherical CLEAs with a biosilica shell were enhanced significantly compared with those of conventional CLEAs. In particular, the spherical CLEAs with a biosilica shell retained 70% of their original activity after 13 cycles, whereas the conventional CLEAs retained only 35% of their original activity. This approach could be an efficient strategy for improving the catalytic properties of CLEAs.Keywords: biomimetic mineralization; CaCO3 microtemplates; immobilization enzyme; spherical CLEAs;

The strategy of light environment control was applied to improve biomass and extracellular polysaccharides (EPS) production of the cyanobacterium Nostoc flagelliforme by adjusting multiple wavelengths (red 660 nm, blue 460 nm, green 520 nm) and light intensity assisted by nitrogen source optimization. A mixed wavelength with low light intensity was more suitable for cell growth. Wavelength shift approach, i.e., manipulation of light wavelength at appropriate culture stages, increased both biomass and EPS production, and optimum shift time was at 9 days. The effects of four nitrogen sources under different light conditions were subsequently evaluated, and urea showed the best performance. The optimized wavelength shift approach (9-day illumination with white light followed by 9-day culture with mixed wavelengths of red/blue/green = 12:5:5) with urea as nitrogen source improved the biomass from 0.72 ± 0.02 to 1.20 ± 0.02 g L−1 (i.e., by 66 %) and EPS production from 27.31 ± 1.00 to 86.65 ± 2.56 mg L−1 (i.e., by 217.3 %). These results provide information on novel culture strategies for microalgal biotechnology by applying light environment control.

•The effects of five light colors on the photosynthetic pigments were studied.•Red light altered the amounts, proportions and location of PC and APC.•Changing red light intensity did not influence the ratio of PC/APC.•Results would help develop the high-efficient N. flagelliforme culture process.Cyanobacteria have evolved a number of photosynthetic strategies by adjusting the properties of the light-harvesting pigments to cope with changes in the light environment. Nostoc flagelliforme is a terrestrial filamentous cyanobacterium that has distinct phycobilisome composition consisting of phycocyanin (PC) and allophycocyanin (APC). The photoregulation process of N. flagelliforme was explored by investigating the effects of different light colors on photosynthetic pigments, which were determined after photoautotrophic growth with red (RL), yellow (YL), green (GL), blue (BL), purple (PL) and white (WL) light of same photosynthetically active photon flux density. Compared with WL, YL, GL, BL and PL changed the amounts of pigments but did not alter the cellular ratio of PC/APC; while RL altered both the amounts and proportions of PC and APC. The observations by confocal laser scanning microscope and transmission electron microscope showed that PC, APC and chlorophyll a were distributed throughout the cells under GL, BL, PL, and WL illumination but located in the outermost layer in RL and YL-grown cells. Further study revealed that changing RL intensity would not influence the ratio of PC/APC but the distribution of pigments shifted from central cytoplasmic region to the periphery of the cells with the increase of intensity. These results implied that N. flagelliforme responded to RL by regulating the composition and location of phycobilisomes and these findings would be of importance to develop the high-efficient process of N. flagelliforme culture.

•EPS monosaccharide composition showed dependence on culture conditions.•The activities of EPS synthesis related enzymes were significantly influenced.•EPS compositions had obvious correlation with FBPase, UGDH, UGPase and PGI.•Correlation under C and N sources conditions was different from light condition.The relationship between monosaccharide composition of Nostoc flagelliforme extracellular polysaccharide (EPS) and activities of EPS synthesis enzymes under various carbon sources, nitrogen sources and light culture condition was investigated. Culture conditions showed significant influences on both monosaccharide composition and related enzyme activities. Under both carbon and nitrogen sources conditions, mannose mole percentage was increased with the increase of initial mole ratio of C/N and positively related to fructose-1, 6-bisphosphatase activity, and glucuronic acid and galactose mole percentages were positively correlated with UDP-glucose dehydrogenase, while arabinose and rhamnose mole percentages were negatively associated with UDP-glucose pyrophosphorylase. Different correlation between monosaccharide composition and enzymes activity from carbon and nitrogen sources conditions was found under light condition. These findings will be helpful to establish a novel fermentation process aimed to produce the N. flagelliforme EPS with desired monosaccharide composition.

Filamentous Nostoc flagelliforme form colloidal complex, with beaded cells interacting with other bacteria embedded in the complex multilayer sheath. However, the species of bacteria in the sheath and the interaction between N. flagelliforme and associated bacteria remain unclear. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of N. flagelliforme from three regions of China. DGGE patterns showed variations in all samples, exhibiting 25 discrete bands with various intensities. The diversity index analysis of bands profiles suggested the high similarity of bacterial communities to each other but also the dependence of microbial composition on each location. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Cyanobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, of which Cyanobacteria was dominant, followed the Proteobacteria. Members of the genus Nostoc were the most abundant in all samples. Rhizobiales and Actinobacteria were identified, whereas, Craurococcus, Caulobacter, Pseudomonas, Terriglobus and Mucilaginibacter were also identified at low levels. Through comparing the bacterial composition of N. flagelliforme from different regions, it was revealed that N. flagelliforme could facilitate the growth of other microorganisms including both autotrophic bacteria and heterotrophic ones and positively contributed to their harsh ecosystems. The results indicated N. flagelliforme played an important role in diversifying the microbial community composition and had potential application in soil desertification.

•Detected higher alcohol and esters of five-hundred samples of commercial Tsingtao fresh beers.•The sensory evaluation was performed by a panel of thirteen judges.•Three multivariate models were established and compared systematically.•Better performance of prediction accuracy was achieved by support vector machine with a radial basis function.•Correlating the potential relationship between sensory evaluation and flavour compounds.Sensory evaluation is regarded as a necessary procedure to ensure a reproducible quality of beer. Meanwhile, high-throughput analytical methods provide a powerful tool to analyse various flavour compounds, such as higher alcohol and ester. In this study, the relationship between flavour compounds and sensory evaluation was established by non-linear models such as partial least squares (PLS), genetic algorithm back-propagation neural network (GA-BP), support vector machine (SVM). It was shown that SVM with a Radial Basis Function (RBF) had a better performance of prediction accuracy for both calibration set (94.3%) and validation set (96.2%) than other models. Relatively lower prediction abilities were observed for GA-BP (52.1%) and PLS (31.7%). In addition, the kernel function of SVM played an essential role of model training when the prediction accuracy of SVM with polynomial kernel function was 32.9%. As a powerful multivariate statistics method, SVM holds great potential to assess beer quality.

Plant-based proteins are valuable supplements to compensate the gap between supply and demand in the food or feed industry. However, they lack essential amino acids, such as lysine in cereal grains and sulfur-containing amino acids in legumes, which greatly limit their wider uses for human and animals. In this study, the contents of nutritional ingredients and antinutritional factors of Caragana korshinskii Kom. and its protein isolates were quantitatively investigated. It was shown that the crude protein contents of C. korshinskii Kom. and its protein isolates obtained by alkaline extraction method (Al-CPI) and acetone precipitation method (A-CPI) were 9.1, 50.1, and 42.6%, respectively. Amino acid contents in C. korshinskii Kom., Al-CPI, and A-CPI basically exceeded the FAO/WHO (2007) reference pattern for adults except sulfur-containing amino acids. The lysine levels in C. korshinskii Kom., Al-CPI, and A-CPI were 4.1, 3.1, and 3.8 mg/100 mg crude protein respectively, which were higher than some other kinds of cereal grains. The methionine in A-CPI (1.39 mg/100 mg crude protein) was even higher than that in soybean. The antinutritional factors in C. korshinskii Kom. and Al-CPI were generally lower than those in some other kinds of legumes except total phenol and tannin. Total phenol and tannins in Al-CPI were 19.02 and 5.66 mg/g dry substance, respectively, but they were undetectable in A-CPI. This study provided a detailed analysis on nutritional and antinutritional factors in C. korshinskii Kom. and its protein isolates, indicating that they have a great potential on food and feed additives.

ε-Poly-l-lysine (ε-PL), a naturally occurring amino acid homopolymer, has been widely used as a food preservative. However, its antimicrobial mechanism has not been fully understood. This study investigated the antimicrobial mode of action of ε-PL on a yeast, Saccharomyces cerevisiae. When treated with ε-PL at the concentration of 500 μg/mL, cell mortality was close to 100% and the phospholipid bilayer curvature, pores, and micelles on the surface of S. cerevisiae were clearly observed by scanning electron microscopy (SEM). At the level of 200 μg/mL, ε-PL significantly inhibited the cell growth of S. cerevisiae. When treated with 50 μg/mL ε-PL, the yeast cell was able to grow but the cell cycle was prolonged. A significant increase in cell membrane permeability was induced by ε-PL at higher concentrations. Metabolomics analysis revealed that the ε-PL stress led to the inhibition of primary metabolic pathways through the suppression of the tricarboxylic acid cycle and glycolysis. It is therefore proposed that the microbiostatic effect of ε-PL at lower levels on S. cerevisiae is achieved by inducing intracellular metabolic imbalance via disruption of cell membrane functions. Moreover, the results suggested that the antimicrobial mechanism of ε-PL on S. cerevisiae can in fact change from microbiostatic to microbicidal when the concentration of ε-PL increased, and the mechanisms of these two modes of action were completely different.

The application of antibiotic treatment with assistance of metabolomic approach in axenic isolation of cyanobacterium Nostoc flagelliforme was investigated. Seven antibiotics were tested at 1–100 mg L−1, and order of tolerance of N. flagelliforme cells was obtained as kanamycin > ampicillin, tetracycline > chloromycetin, gentamicin > spectinomycin > streptomycin. Four antibiotics were selected based on differences in antibiotic sensitivity of N. flagelliforme and associated bacteria, and their effects on N. flagelliforme cells including the changes of metabolic activity with antibiotics and the metabolic recovery after removal were assessed by a metabolomic approach based on gas chromatography–mass spectrometry combined with multivariate analysis. The results showed that antibiotic treatment had affected cell metabolism as antibiotics treated cells were metabolically distinct from control cells, but the metabolic activity would be recovered via eliminating antibiotics and the sequence of metabolic recovery time needed was spectinomycin, gentamicin > ampicillin > kanamycin. The procedures of antibiotic treatment have been accordingly optimized as a consecutive treatment starting with spectinomycin, then gentamicin, ampicillin and lastly kanamycin, and proved to be highly effective in eliminating the bacteria as examined by agar plating method and light microscope examination. Our work presented a strategy to obtain axenic culture of N. flagelliforme and provided a method for evaluating and optimizing cyanobacteria purification process through diagnosing target species cellular state.

The emulsifying, flocculating, and physicochemical properties of purified exopolysaccharide (EPS) of terrestrial cyanobacterium Nostoc flagelliforme cultured in liquid media were investigated. The EPS was defined as heteropolysaccharide composed by 41.2 % glucose, 21.1 % galactose, 21.0 % mannose, 2.5 % fructose, 3.6 % ribose, 1.7 % xylose, 0.6 % arabinose, 3.0 % rhamnose, 0.9 % fucose, and 4.3 % glucuronic acid. The EPS possessed higher intrinsic viscosity than other cyanobacterial strains as reported and displayed pseudoplastic behavior in aqueous solution. The EPS produced more stable emulsions with tested hydrocarbons and oils than xanthan gum, and the emulsification indexes with n-hexadecane, liquid paraffin, and peanut oil were higher than 50 %, indicating the strong emulsion-stabilizing capacity. The EPS showed peak flocculating rates of 93.5 and 86.1 % in kaolin and MgO suspension, respectively, and exhibited a better flocculation performance than Al2(SO4)3 and xanthan gum. These results demonstrated that the EPS of N. flagelliforme was a very promising candidate for numerous industrial applications, as it had higher intrinsic viscosity, good emulsification activity, and excellent flocculation capability.

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.

Dissociated cells separated from a natural colony of Nostoc flagelliforme were cultivated heterotrophically in the darkness on glucose under fed-batch culture conditions. The effects of carbon sources (glucose, fructose, xylose, and sucrose) and concentrations on cell growth and extracellular polysaccharide (EPS) production were investigated. At harvest, the culture contained 1.325 g L−1 of biomass and 117.2 mg L−1 of EPS, respectively. The gravimetric EPS production rate was 16.7 mg g−1 cell dry weight day−1, which was 2.1 times higher than previously reported. Using sigmoid model, batch fermentation of N. flagelliforme was kinetically simulated to obtain equations including substrate consumption, biomass growth, and EPS accumulation. Results from a simulation correlated well with the experimental ones, indicating that this method could be useful in studying EPS production from batch and fed-batch cultures.

Seeking cheap, sustainable protein sources greatly facilitates in alleviating the dependence upon expensive animal-based protein in many developing countries. Caragana korshinskii Kom. offers a good alternative feedstock because of its high-content of protein, low fertilizer and pesticide requirements, excellent stress (high salty and less water) tolerance, wide adaptability, etc. The functional properties of C. korshinskii Kom. protein isolates by three different extraction methods were investigated. The extraction processes greatly influenced the physiological characteristics of protein isolates. C. korshinskii Kom. protein isolate by traditional alkaline extraction (Al-CPI) exhibited good performance on emulsifying activity index, oil and water absorption capacity, and foaming property compared to A-CPI (C. korshinskii Kom. protein isolate by the acetone precipitation method) and TCA-CPI (C. korshinskii Kom. protein isolate by trichloroacetic acid–acetone precipitation). The water and oil adsorption capacities of Al-CPI were observed at 4.99 and 3.45 g/g, respectively, even much higher than those of soy protein isolate (SPI) (3.94 and 2.95 g/g, respectively). The highest foaming capacity was observed by Al-CPI at 185.0%, followed by A-CPI (177.5%), TCA-CPI (142.5%), and SPI (141.9%), respectively. It has to be noted that A-CPI showed good solubility at acidic pH and excellent in vitro digestibility. After sequential pepsin–trypsin digestion, the percentage of N release of A-CPI reached up to 83.7%, which was 1.63 times that of Al-CPI (51.2%), 1.19 times that of TCA-CPI (70.1%), and slightly higher than that of the commercial SPI (82.5%). These results indicate that C. korshinskii Kom. holds great potential for application in the animal feed and food additive industry.

ɛ-Poly-L-lysine (ɛ-PL) is an L-lysine linear homopolymer, which is produced by bacteria belonging to the Streptomycetaceae family and by ergot fungi. However, the production of ɛ-PL by the wild bacteria strain is very low, which limits its utilization. In most bacteria including the Streptomyces genus, L-lysine is a precursor of ɛ-PL and is biosynthesized by the L-aspartate pathway. Aspartokinase (Ask) is the first key enzyme in this pathway and is subject to complex regulation such as the feedback inhibition by the end product amino acids. In addition, phosphoenolpyruvate carboxykinase is feedback-regulated by L-aspartate. To reduce these feedback inhibitions and to improve ɛ-PL productivity, resistant mutants were produced using sulfaguanidine (SG) + glycine + L-lysine + DL-3-hydroxynorvaline (AHV) as selective markers. Using the interaction between ɛ-PL and the charged dye in the solid culture medium, hundreds of colonies were simultaneously screened in a quick and effective manner. Finally, one ɛ-PL-producing strain, Streptomyces diastatochromogenes L9, was selected. The productivity of this strain during flask fermentation was 0.77 g/L, which was 15% higher than that of the original strain. Moreover, its fermentation performance and genetic characteristics were very stable.

Nostoc flagelliforme in liquid culture was cultivated on sand bed materials as inoculum and formed a biological crust. The biological crust had no common appearance of wild N. flagelliforme thallus in naked eye observation, but it had natural morphology of wild N. flagelliforme thallus in microscopic observation. By examining the photosynthetic activity, N. flagelliforme cells on sand were found to be heat resistant. The sand grain size had some influence on N. flagelliforme growth, and the growth rate on fine sand was higher than on coarse sand. It is necessary to consider the characteristics of sand particle size to prevent desertification. N. flagelliforme cells could form the algal biological crust, which indicates that the biological crust has potential application in soil desertification and was extremely important to improve desertification.

Nostoc flagelliforme was cultivated on three solid bed-materials which have different wettability. The results show that the wettability of solid bed-materials is very important to the need for water in N. flagelliforme, and also greatly affects photosynthetic activity, polysaccharide secretion and the morphology of the cells. Glass residue has the best wettability, followed by sand, and then nylon6 (PA6). We show that only sand is suitable for the growth of N. flagelliforme. When cultivated on sand for 100 days, the cells maintained a typical rosary-like shape. On glass residue, two to three vegetative cells formed a group with slime. On PA6, cells were in the form of loose group with slimes. N. flagelliforme is applicable to desert control as it cannot only grow on the sand, but also adhere to sand to form a biological crust.

The feasibility of a novel bacteriostatic sausage casing made of bacterial cellulose (BC) embedded with ɛ-polylysine (ɛ-PL) was evaluated. The ɛ-PL/BC composite was prepared by immersing BC tubes in ɛ-PL solution and its characteristics were analyzed with Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and atomic force microscopy (AFM) techniques, respectively. The results suggested that ɛ-PL molecules were incorporated into the cellulose fiber networks and the ɛ-PL/BC composite might have a novel unique structure. No significant loss of antimicrobial activity was observed even after autoclaving at 121°C for 30 min and the oxygen permeability was far below than that of polyethylene (PE) and polyvinyl alcohol (PVA) membrane. Its tensile strength was 51.8 MPa. The ɛ-PL/BC composite exhibits bacteriostatic and/or bacteriocidal activities against a broad spectrum of Gram-positive and Gram-negative bacteria; as a result, an extended shelf life than controls was observed for sausage packaged with the ɛ-PL/BC composite.

ɛ-Poly-l-lysine (ɛ-PLL) produced by Streptomyces albulus CGMCC 1986 was fractionated using ultra-filtration technique with 2 and 5 kDa cut-offs of membrane. The number-average molecular weight of each fraction was determined by 1H nuclear magnetic resonance (NMR) method. The number-average molecular weights of the cutoffs of 5 and 2 kDa and the filtrate are 4,230.95, 3,687.80, and 1,900.82 Da, respectively. 1H NMR indicates the chemical shifts of α-H, β-H, γ-H, δ-H, and ɛ-H are very similar to all the fractions. Fourier transform-infrared (FTIR) spectra showed that the ɛ-PLL solid samples obtained by freeze-drying at pH 5 with molecular weights higher than 2 kDa take on a β-turn conformation, however, the fraction with molecular weight smaller than 2 kDa adopts random coil structure. The antibacterial test proved that the fraction between 2 and 5 kDa of membranes behaves the highest antibacterial activity than other fractions for the test strains of Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, and Shigella.

Bacterial cellulose has been found to be attractive as a novel scaffold material due to its unique material properties. Porosity is the most important morphological parameter in the design of scaffolds for tissue engineering. The effects of fermentation conditions (cultivation time and inoculation volume) and post-treatment methods (alkali treatment and drying methods) on the porosities of bacterial cellulose membranes were investigated. With extended cultivation time and increased inoculation volume, more micro-fibrils were secreted by bacteria, which resulted in a more compact structure and diminished porosity. The porosities of alkali-treated bacterial cellulose membranes was in the order of K2CO3 > Na2CO3 > KOH > NaOH. Freeze-dried membranes had much higher porosity (92%) than the hot air-dried ones (65%). The experimental results suggested that bacterial cellulose with controlled porosities could be prepared by varying fermentation conditions and post-treatment methods. The resulting bacterial cellulose is regarded as a scaffold material of great potentialities.

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L−1 cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.

Noscocflagelliforme is a terrestrial macroscopic cyanobacterium with high economic value. Free-living cells that were separated from a natural colony of N. flagelliforme were cultivated in a 20-L photobioreactor for 16 days at five agitation rates with impeller tip speeds at 0.3, 0., 0.8, 1.0, and 1.5 m·s−1. With different impeller tip speeds there were significant differences in the cell growth and polysaccharide production, and different types of cell colonies appeared because of different shear forces caused by agitation. At harvest time, cell concentrations with tip speeds of 0.8 and 1.0 m·s−1 were clearly higher than those with the other three tip speeds, but dry cell weights with the tip speeds of 0.3, 0.5, 0.8, and 1.0 m·s−1 were almost the same. The highest RPS (polysaccharide that released into liquid medium) production was obtained with the tip speeds of 0.8 and 1.0 m·s−1, while the highest EPS (polysaccharide that formed capsule or slime layer) production was obtained with the tip speed of 0.5 m·s−1. The tip speed of 1.5 m·s−1 was harmful for both cell growth and polysaccharide production, indicating that an appropriate shear force was needed in the liquid suspension culture of N. flagelliforme.

Nostoc flagelliforme cells were studied with regard to the physico-chemical characterization of the extracellular polysaccharides (EPS) secreted in a liquid suspension culture. The hydrolyzed EPS were determined to be composed of four neutral sugars, which were glucose (43.2%), xylose (20.6%), galactose (29.9%), and mannose (6.3%). The glucuronic acid was the only uronic acid identified in the residue. The apparent molecular weight was estimated at 2.79×105. The Fourier transform infrared spectra showed that the EPS evidenced characteristics typical of non-sulfated polysaccharides. The UV spectrum and Bradford reaction indicated that there were no nucleic acids and proteins in them. The thermal analysis showed a decomposition peak at 245°C on the thermogravimetric (TG) curves. The scanning electron microscope (SEM) analysis indicated that the EPS possessed a porous structure. The observed microstructural irregularities indicated that the polysaccharide was a type of amorphous solid. These results showed that the EPS ofN. flagelliforme cells might be ernployed as a substitute for those normally derived from field colonies. The results of this study may prove to be beneficial to the protection of the natural resource represented byN. flagelliforme.

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing PLPR promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.