A temperature-induced chitosanase bacterial cell-surface display system for the efficient production of chitooligosaccharides.

OBJECTIVE: To establish a temperature-induced chitosanase bacterial cell-surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications. RESULTS: Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A (1 copy) and 2InaQ-N-CSN46A (2 copies). The specific enzyme activity of 2InaQ-N-CSN46A reached 761.34 ± 0.78 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in the 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. Gel permeation chromatography revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 °C. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs. CONCLUSIONS: In this study, chitosanase was expressed on the surfaces of E. coli by increasing the induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provides a novel strategy for industrial COS production.

Bacterial Cellulose-Alginate Composite Beads as Yarrowia lipolytica Cell Carriers for Lactone Production.

The demand for natural lactone gamma-decalactone (GDL) has increased in the fields of food and cosmetic products. However, low productivity during bioprocessing limits its industrial production. In this study, a novel composite porous cell carrier, bacterial cellulose-alginate (BC-ALG), was used for long-term biotransformation and production of GDL. The effects of this carrier on biotransformation and related mechanisms were investigated. BC-ALG carriers showed improved mechanical strength over ALG carriers, with their internal embedded cell pattern changed to an interconnected porous structure. In five repeated-batch biotransformation experiments, the maximum concentration of GDL obtained in culture with BC-ALG carriers was 8.37 g/L, approximately 3.7 times higher than that from the medium with an ALG carrier alone. The result indicated that multiple hydrogen bonding interactions at the interface between BC and ALG contributed to the compatibility and stability of BC-ALG carriers. On the basis of the above results, the BC-ALG composite carrier can be considered ideal for immobilisation of cells for the production of GDL on an industrial scale, and has the potential to be utilised in other biological processes.

Low molecular weight chitosan oligosaccharides form stable complexes with human lactoferrin.

Proteins in tears, including human lactoferrin (HLF), can be deposited and denatured on contact lenses, increasing the risk of microbial cell attachment to the lens and ocular complications. The surfactants currently used in commercial contact lens care solutions have low clearance ability for tear proteins. Chitosan oligosaccharide (COS) binds to a variety of proteins and has potential for use in protein removal, especially in contact lens care solutions. Here, we analyzed the interaction mechanism of COSs hydrolyzed from chitosan from different resources with HLF. The molecular weights (MWs) and concentrations of COSs were key factors for the formation of COS-HLF complexes. Lower MWs of COSs could form more stable COS-HLF complexes. COS from Aspergillus ochraceus had a superior effect on HLF compared with COS from shrimp and crab shell with the same MWs. In conclusion, COSs could bind to and cause a conformational change in HLF. Therefore, COSs, especially those with low MWs, have potential as deproteinizing agents in contact lens care solution.

Human Papillomavirus E1 Protein Regulates Gene Expression in Cells Involved in Immune Response.

Human papillomavirus belongs to papovaviridae family papillomavirus A, a spherical deoxyribonucleic acid (DNA) virus, which can cause the proliferation of squamous epithelial cells of human skin or mucous membranes. With the rapid increase in the incidence of condyloma acuminatum among STDs and the increase in diseases caused by HPV infection, HPV infection has seriously endangered human health. In this paper, the in vitro detection of HPV E1 protein was realized using AgNCs-dsDNA. And through the test of this detection method, we calculated that the detection limit of this method is 0.886 nM. Compared with other methods for detecting E1 protein in vitro, this method has high sensitivity and simple operation. In addition, the detection method also has good anti-interference and selectivity, and can realize the detection of E1 in serum samples. The transfection efficiency of BLV-miR-B4-3p mimics at different time points was determined by quantitative real-time PCR (qPCR); the transcriptome sequencing of lymphocytes transfected with different concentrations of BLV-miR-B4-3p mimics was performed, and differential gene clustering was performed on the sequencing results. And the BLV-miR-B4-3p target gene prediction and transcriptome analysis results were verified by qPCR. The effects of BLV-miR-B4-3p on the transcriptional levels of immune-related cytokines in human lymphocytes were analyzed. Transcriptome sequencing analysis showed that after BLV-miR-B4-3p entered lymphocytes, a total of 556 differentially expressed genes were obtained. GO enrichment and KEGG analysis results showed that BLV-miR-B4-3p could independently activate influenza. The signaling pathway ultimately affects the body's immune system process, stress response, defense response, immune response, and other biological processes. After BLV-miR-B4-3p enters lymphocytes, it will lead to abnormal lymphocyte immune function, including the mRNA expression of TNF-α in Th1 cytokines which was significantly increased (P < 0.05), and the expression of IL-10 in Th2 cytokines was significantly increased (P < 0.05). The mRNA expression was significantly decreased (P < 0.05), and the mRNA expression of IL-27 was significantly increased (P < 0.001), which did not affect the mRNA expression of lymphocyte proliferation and activation-related regulators. The tumor suppressor breast cancer 1 (BRCA1) and antimicrobial peptide CAMP were significantly increased, and decreased (P < 0.001), and the expression of pro-apoptotic factor Caspase9 showed a significant downward trend (P < 0.05).

Characterization of 405B8H3(D-E), a newly engineered high affinity chimeric LAG-3 antibody with potent antitumor activity.

Lymphocyte activation gene-3 (LAG-3) is a type I transmembrane protein with structural similarities to CD4. Overexpression of LAG-3 enables cancer cells to escape immune surveillance, while its blockade reinvigorates exhausted T cells and strengthens anti-infection immunity. Blockade of LAG-3 may have antitumor effects. Here, we generated a novel anti-LAG-3 chimeric antibody, 405B8H3(D-E), through hybridoma technology from monoclonal antibodies produced in mice. The heavy-chain variable region of the selected mouse antibody was grafted onto a human IgG4 scaffold, while a modified light-chain variable region was coupled to the human kappa light-chain constant region. 405B8H3(D-E) could effectively bind LAG-3-expressing HEK293 cells. Moreover, it could bind cynomolgus monkey (cyno) LAG-3 expressed on HEK293 cells with a higher affinity than the reference anti-LAG-3 antibody BMS-986016. Furthermore, 405B8H3(D-E) promoted interleukin-2 secretion and was able to block the interactions of LAG-3 with liver sinusoidal endothelial cell lectin and major histocompatibility complex II molecules. Finally, 405B8H3(D-E) combined with anti-mPD-1-antibody showed effective therapeutic potential in the MC38 tumor mouse model. Therefore, 405B8H3(D-E) is likely to be a promising candidate therapeutic antibody for immunotherapy.

A Broad-Range Disposable Electrochemical Biosensor Based on Screen-Printed Carbon Electrodes for Detection of Human Noroviruses.

Human noroviruses (HuNoVs) are the major non-bacterial pathogens causing acute gastroenteritis in people of all ages worldwide. No stable culture system in vitro is available for routing the detection of multiple strains of HuNoVs. A simple and rapid method for detection of HuNoVs is of great significance for preventing and controlling this pathogen. In this work, an electrochemical biosensor for sensitive and fast detection of HuNoVs was constructed based on a screen-printed carbon electrode (SPCE). Gold nanoparticles and protein-A were applied on the SPCE surface for enhancement of the electrical signals and the linkage of antibodies with a fixed orientation, respectively. A monoclonal antibody (MAb) against the S domain protein of the viral capsid (VP1) was further immobilized on the SPCE to bind HuNoVs specifically. The binding of VP1 to the coated MAbs resulted in the reduction of conductivity (current) measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The reduction in the current was correlated to the concentration of VP1/HuNoVs. The detection limitation of Genogroup I.1 (GI.1) VP1 and Genogroup II.4 (GII.4) VP1 was 0.37 ng/ml (≈1.93×107 HuNoVs/mL) and 0.22 ng/ml (≈1.15×107 HuNoVs/mL), respectively. The detection limitation of both GI and GII HuNoVs in clinical fecal samples was 104 genomic copies/mL. The results could be obtained in 1 h. We demonstrated that this disposable electrochemical biosensor was a good candidate for rapid detection of different genogroup and genotype HuNoVs.

Enhanced biotransformation of 2-phenylethanol with ethanol oxidation in a solid-liquid two-phase system by active dry yeast.

2-Phenylethanol (2-PE) can be produced from L: -phenylalanine (L: -Phe) with the oxidation degradation of ethanol by active dry yeast. In this study, the catalysis effect of ethanol on biotransforming L: -Phe into 2-PE by yeast was evaluated and optimized. The results indicated that increasing ethanol concentration was beneficial for enhancing 2-PE concentration but lowered the 2-PE productivity. Initial ethanol concentration above 25 g/l could strongly inhibit the 2-PE production. To obtain 2-PE with desirable concentrations with an economical operation mode, three fed-batch biotransformation operation methods using ethanol or/and glucose were carried out in a solid-liquid two-phase system. When using ethanol alone with the initial concentration of 10 g/l, the total concentration and overall productivity of 2-PE were 7.6 g/l and 0.065 g l(-1) h(-1), respectively. Furthermore, an experiment with controlled glucose solely (higher than 2 g/l) was finished. In this case, phenylacetaldehyde (PA) was detected along with ethanol accumulation, suggesting that reaction of PA → 2-PE in Ehrlich pathway was inhibited. To further enhance 2-PE production by using glucose only, a novel operation strategy to simultaneously control rates of glucose glycolysis and ethanol oxidative degradation with the aid of ISPR techniques was developed. With this strategy, 2-PE concentration and yield based on glucose consumption reached a higher level of 14.8 g/l and 0.12 g-PE/g-glucose, respectively, and these are the highest values reported up to date with the fed-batch biotransformation operation mode.

Effects of Impeller Geometry on the 11α-Hydroxylation of Canrenone in Rushton Turbine-Stirred Tanks.

The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments using a 50 mm four-blade impeller showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s-1 and 35.31 s-1, respectively. We therefore concluded that appropriate shear should be applied in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.

Advances and Future Perspective on Detection Technology of Human Norovirus.

Human norovirus (HuNoV) is a food-borne pathogen that causes acute gastroenteritis in people of all ages worldwide. However, no approved vaccines and antiviral drugs are available at present. Therefore, the development of accurate and rapid detection technologies is important in controlling the outbreak of HuNoVs. This paper reviewed the research progress on HuNoV detection, including immunological methods, molecular detection and biosensor technology. Immunological methods and molecular detection technologies are still widely used for HuNoV detection. Furthermore, biosensors will become an emerging developmental direction for the rapid detection of HuNoVs because of their high sensitivity, low cost, easy operation and suitability for onsite detection.

Improved 11α-hydroxycanrenone production by modification of cytochrome P450 monooxygenase gene in Aspergillus ochraceus.

Eplerenone is a drug that protects the cardiovascular system. 11α-Hydroxycanrenone is a key intermediate in eplerenone synthesis. We found that although the cytochrome P450 (CYP) enzyme system in Aspergillus ochraceus strain MF018 could catalyse the conversion of canrenone to 11α-hydroxycanrenone, its biocatalytic efficiency is low. To improve the efficiency of 11α-hydroxycanrenone production, the CYP monooxygenase-coding gene of MF018 was predicted and cloned based on whole-genome sequencing results. A recombinant A. ochraceus strain MF010 with the high expression of CYP monooxygenase was then obtained through homologous recombination. The biocatalytic rate of this recombinant strain reached 93 % at 60 h without the addition of organic solvents or surfactants and was 17-18 % higher than that of the MF018 strain. Moreover, the biocatalytic time of the MF010 strain was reduced by more than 30 h compared with that of the MF018 strain. These results show that the recombinant A. ochraceus strain MF010 can overcome the limitation of substrate biocatalytic efficiency and thus holds a high poten tial for application in the industrial production of eplerenone.

Efficient synthesis of naphtho[1,2-e][1,3]oxazine derivatives via a chemoselective reaction with the aid of low-valent titanium reagent.

A series of new naphtho[1,2-e][1,3]oxazine derivatives such as trans-1,3-diaryl-1H-naphtho[1,2-e][1,3]oxazine-2(3H)-carbonyl chloride, 1-aryl-2-benzyl-1,2- dihydronaphtho[1,2-e][1,3]oxazine-3-one, and trans-1,3-diaryl-1H-naphtho[1,2-e] [1,3]oxazine-2(3H)-carbaldehyde were selectively synthesized via a chemoselective reaction of 1,3-diaryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazines and triphosgene or triethyl orthoformate, respectively, induced by different low-valent titanium systems. This method has the advantages of short reaction time (15 min), convenient manipulation, and high chemoselectivity.

Biotransformation of 12-hydroxystearic acid to gamma-decalactone: Comparison of two separation systems.

Biotransformation of natural products to the natural flavoring, gamma-decalactone (GDL), has attracted considerable attention. However, improving its yield is challenging due to its high feedback inhibition of yeast cells, which lowers the productivity of the biotransformation process. In this study, we compared two in situ separation processes established by adding either resin (HZ-816) or cyclopentasiloxane (DC345) to a biotransformation medium and investigated their efficiency and effect on yeast metabolism. Compared with a control, yields from the medium with HZ-816 and DC345 increased by 140% and 175%, respectively. However, after 84 h of biotransformation, the protein leakage in the medium with HZ-816 and DC345 was respectively 2.04 times and 1.43 times that of the control. Meanwhile, the mortality of yeast cells was 32.8% and 24.0% in the medium with HZ-816 and DC345, respectively, whereas that in the control was 20.1%. Our findings indicate that a cyclase is involved in the final step of the biotransformation. The activity of the yeast cyclase in the DC345 system was 3.33 times greater than that in the HZ-816 system. The DC345 system was superior to the HZ-816 resin system in this separation process because its yield was 30.8% greater and it had less cellular damage. Thus, we showed that the DC345 system has potential as a new separation system for the production of GDL by biotransformation.

Whole-Genome Sequence of Enterobacter sp. Strain MF024, Isolated from Soil in Shanghai, China.

We report here the draft genome sequence of Enterobacter sp. strain MF024, a bacterium that can biosynthesize 2-phenylethanol through both the Ehrlich pathway and a de novo pathway. It has potential use for the production of 2-phenylethanol.

Enhanced Biotransformation Productivity of Gamma-Decalactone from Ricinoleic Acid Based on the Expanded Vermiculite Delivery System.

Natural gamma-decalactone (GDL) produced by biotransformation is an essential food additive with a peach-like aroma. However, the difficulty of effectively controlling the concentration of the substrate ricinoleic acid (RA) in water limits the biotransformation productivity, which is a bottleneck for industrialization. In this study, expanded vermiculite (E-V) was utilized as a carrier of RA to increase its distribution in the medium. E-V and three commonly used organic compounds were compared with respect to their effects on the biotransformation process, and the mechanism was revealed. Scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis indicated that RA was physically adsorbed onto the surface of and inside E-V instead of undergoing a chemical reaction, which increased the opportunity for interactions between microorganisms and the substrate. The highest concentration of GDL obtained in the medium with E-V was 6.2 g/l, which was 50% higher than that in the reference sample. In addition, the presence of E-V had no negative effect on the viability of the microorganisms. This study provides a new method for producing natural GDL through biotransformation on an industrial scale.

Characterization of conditions for bacteria-human norovirus capsid P protein complex (BPC) binding to and removal from Romaine lettuce extract.

Norovirus is a very contagious virus that causes acute gastroenteritis. Contaminated produce is a main vehicle for dissemination of human noroviruses (HuNoVs). As HuNoVs could bind to bacteria effectively, it is highly possible that produce could be contaminated by bacteria-HuNoVs complex. In this study, we used a bacterial-surface-display system to express genogroup I (GI) or genogroup II (GII) HuNoV capsid protein (P protein) on the surface of bacteria. The bacteria-P protein complex (BPC) was used to characterize the conditions for binding to Romaine lettuce extract and removal of the bound BPCs. We demonstrated both GI and GII BPCs could bind to extract from leaf (LE) and vein (VE) effectively. Carbohydrates in LE and VE were involved in GI BPCs binding, and both carbohydrates and proteins were involved in GII BPCs binding. Saliva from both type A and O secretors could completely block binding of both BPCs to LE and VE. Saliva from type B secretors only partially blocked binding of GII but not GI BPCs to LE and VE. However, LE- and VE-bound BPCs could not be reversely removed by washing solution containing free HBGAs from saliva. The binding of GI BPCs to LE and VE was enhanced when pH was below pI (6.1) of GI and reduced when pH was above pI of GI (p < 0.05). The optimal binding for GII BPCs to LE and VE occurred at pI (6.4) of GII. All LE- or VE-bound BPCs could be reversely removed by washing with low (3.0-5.0) or high (9.0-10.0) pH buffer. The effect of ionic strength (NaCl and MgCl2, from zero to 100 g/L) on binding of BPCs to LE and VE was tested. The optimal ionic strength for binding of BPCs to LE and VE was 10.0 g/L (GI) and 5.0 g/L (GII) for NaCl, and 5.0 g/L for MgCl2. LE- and VE-bound BPCs could be reversely removed by washing with high ionic solutions. All LE- or VE- bound BPCs could be released when washed with NaCl concentrations of above 75.0 g/L (GI) and 25.0 g/L (GII), or with MgCl2 concentrations of above 75.0 g/L (GI) and 50.0 g/L (GII). Binding of BPCs to LE and VE was inhibited in the presence of Tween-80 (nonionic surfactant) as low as 0.05% (v/v). All LE- and VE-bound BPCs could be reversed by Tween-80 concentrations over 0.1% (v/v). The study provided important parameters for BPCs binding to and removal from lettuce extract.

Bacterial Surface-Displayed GII.4 Human Norovirus Capsid Proteins Bound to HBGA-Like Molecules in Romaine Lettuce.

Human Noroviruses (HuNoVs) are the main cause of non-bacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein on bacterial surface and used it as a new strategy to explore interaction between HuNoV protein and receptor candidates from romaine lettuce. The surface-displayed HuNoV proteins were confirmed on the surface of the transformed bacteria by an immunofluorescence assay. The distribution patterns of the surface-displayed HuNoV proteins in romaine lettuce were identified through a confocal immunofluorescence assay. The surface-displayed HuNoV proteins could be found in the stomata, and the surfaces of vein and leaf of romaine lettuce. The surface-displayed HuNoV proteins could be captured by an ELISA assay utilizing extract from leaf (LE) or vein (VE). The binding of the surface-displayed HuNoV proteins to LE or VE could be competitively blocked by histo-blood group antigens from human saliva. In addition, the binding of the surface-displayed HuNoV proteins to LE or VE could also be attenuated by heat denaturation of lettuce proteins, and abolished by oxidation of lettuce carbohydrates. The results indicated that histo-blood group antigen-like molecules in LE or VE were involved in the binding of the surface-displayed HuNoV proteins to romaine lettuce. All data demonstrated that the surface-displayed HuNoV proteins could be utilized in a new and simple system for investigation of the interaction between the HuNoVs and their candidate ligands.

[The product identification study on the isolating process of strains produced trehalose].

In the isolating process of stains whose endocellular enzymes can produce trehalose on starch or maltooligosaccharides, we discovered the components of enzymatic reactant were complicated and it was difficult to purify them each other, however, we have to know quickly whether there was tehalose in the enzymatic reactant above. In order to make it clearly, thinner layer chromatography, high performance liquid chromatography-electrospray ionization mass spectrometry and 13C-nuclear magnetic resonance were adopted. The unknown oligosaccharide produced by Arthrobacter nicotinovorus D-97 was identified rapidly and this unknown oligosaccharide needn't be purified from the enzymatic reactant solely. The production on starch or maltooligosaccharides by endocellular enzymes of Arthrobacter nicotinovorus D-97 is trehalose. This research method in our paper can be applied in the isolating process of production trehalose and other functional glucobioses strains.

Effects of aroma quality on the biotransformation of natural 2-phenylethanol produced using ascorbic acid

Background Natural 2-phenylethanol (2-PE) is an important flavoring that emits the aroma of roses. During biotransformation, the aroma quality of natural 2-PE is affected by its main by-products, which include butanol, isobutyric acid, butyric acid, and isovaleric acid. Thus, controlling undesirable by-product formation can reduce the effect of odor on 2-PE aroma quality. Results 2-PE was produced through biotransformation using l-phenylalanine as a substrate and glucose as a carbon source. Ascorbic acid was added to the system to improve the redox reaction and suppress the generation of by-products. Principal component analysis of the aroma quality of 2-PE was performed using an electronic nose. Similarity analysis revealed that the effects of four by-products on 2-PE aroma quality may be ranked in the following order: isovaleric acid > butyric acid > isobutyric acid > butanol. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Conclusions 2-PE produced through the addition of ascorbic acid exhibited the closest aroma similarity to the standard 2-PE sample.

[Study on the production of trehalose by bacterium D-97 endocellular enzymes using HPLC/RI and HPLC/ESI-MS].

The mechanism in which trehalose is produced from dextrin or starch hydrolyzate by endocellular enzymes of bacterium D-97 can be elucidated high performance liquid chromatography (HPLC) with differential refraction detection (RI) basically, including the effect of the different carbon sources on the endocellular trehalose-producing enzymes in bacterium D-97 and the possibility or ability of the endocellular enzymes to produce trehalose using maltooligosaccharides of different chain lengths. After purification of endocellular enzymes of bacterium D-97, two enzymes (called Enzyme A and Enzyme B) related to trehalose synthesis were found. The unknown oligosaccharides produced by Enzyme A were analyzed with the HPLC/RI and HPLC/ESI-MS (electrospray ionization mass spectrometry). The results showed the relative molecular masses of the unknown oligosaccharides were the same as those of the enzymatic reaction substances (maltotriose, maltotetraose and maltopentaose) respectively. In combining with other results of biological experiments, these unknown oligosaccharides had been identified basically. There was no reduction power in these unknown oligosaccharides and only one trehalose residue exited in the molecular chain of these unknown oligosaccharides.