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Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from Aspergillus niger cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of A. niger in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that A. niger could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 U ml−1 and 169.88 ± 11.05 U ml−1, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from A. niger using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications. [ABSTRACT FROM AUTHOR]

Thermophilic methane fermentation was a valid approach for handling the stillage eluted from ethanol fermentation of waste paper and kitchen waste. The wide organic loading rate (OLR) range (2–14 g VTS/(L d)) for stable performance and relatively high energy recovery efficiency (79.0%) were achieved, and OLR of 8 g VTS/(L d) was optimum for achievement of highest biogas evolution and VTS removal efficiency. Microbial community analysis revealed that hydrolysis of cellulose was the critical step for methane production from the stillage. [ABSTRACT FROM AUTHOR]

Due to the presence of antibiotics in environmental water and their potential influence on the occurrence of antibiotic-resistant bacteria, development of a detection method suitable for the screening of environmental water for antibiotics is required. In this study, we developed a simple colorimetric paper-based biosensor based on a novel principle for the detection of antibiotics inhibiting bacterial protein synthesis, including aminoglycosides, tetracycline, chloramphenicol, and macrolides. This biosensor is based on the detection of a color change induced by β-galactosidase, which is synthesized on freeze-dried paper discs containing an in vitro transcription/translation system. When a water sample without antibiotics is applied to the paper discs, β-galactosidase can be synthesized, and it hydrolyzes a colorimetric substrate, resulting in a color change from yellow to purple. By contrast, in the presence of antibiotics, the color change can be hampered due to an inhibition of β-galactosidase synthesis. We investigated the effect of the incubation temperature and pH of water samples and confirmed that the paper discs showed the color change to purple in the ranges of 15–37°C and pH 6–10. We observed concentration-dependent color variations of the paper discs by the naked eye and further estimated detection limits to be 0.5, 2.1, 0.8, and 6.1 μg/mL for paromomycin, tetracycline, chloramphenicol, and erythromycin, respectively, using digitized pictures. The paper-based biosensor proved to detect 0.5 μg/mL paromomycin, spiked in real environmental water samples, by the naked eye. [ABSTRACT FROM AUTHOR]

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10–100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics. [ABSTRACT FROM AUTHOR]

Abstract: The production of hemicellulases by Thermomyces lanuginosus SK using oatspelts xylan was examined during submerged cultivation. A high level of extracellular xylanase (346±10 U ml−1) production was observed on the fifth day; however, accessory enzyme levels were low. T. lanuginosus SK was further subjected to UV and N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. The T. lanuginosus MC 134 mutant showed a 1.5 fold increase in xylanase production on oatspelts xylan, compared to the wild type strain. Xylanase production was further enhanced to 3299±95 U ml−1 by using corn cobs under optimized growth conditions. A reduction in xylanase production was observed in a 5 L fermenter. Also, the biobleaching efficiency of crude xylanase was evaluated on bagasse pulp, and a brightness of 46.07±0.05% was observed with the use of 50 U of crude xylanase per gram of pulp. This brightness was 3.6 points higher than that of the untreated samples. Reducing sugars (25.78±0.14 mg g−1) and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. T. lanuginosus MC 134 has a potential application in the pulp and paper industries. [Copyright &y& Elsevier]

Trichoderma harzianum EU2-77 was a mutant strain of the wild-type strain T. harzianum NP13a isolated in Singapore. A multi-mutagenesis one-screening (MMOS) method was developed to further improve strain EU2-77 and a new mutant EUA20 was obtained. It exhibited filter paper cellulase (FPase) activity up to 14.79 IU/mL within 6 days shake flask cultivation. Activities of FPase, endoglucanase, β-glucosidase, and xylanase, and protein content by EUA20 were respectively increased to 5.73, 4.35, 7.34, 1.80 and 2.70 folds. Using pretreated oil palm empty fruit bunch (OPEFB) and corncob powder as the substrates, strain EUA20 presented approximate 6.52 and 8.80 IU/ml FPase activity. Reducing sugar yield of 615.8 and 636.8 mg/g biomass were respectively obtained for OPEFB and corncob powder using cellulolytic enzymes of strain EUA20. Our results demonstrated that mutant strain EUA20 had great potential in on-site cellulase production for effective biomass bioconversion. [ABSTRACT FROM AUTHOR]

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter , one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus , Clostridium , and Caldicellulosiruptor. Calicellulosiruptor , which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen. [ABSTRACT FROM AUTHOR]

To achieve the high-level stable expression of chlorothalonil hydrolytic dehalogenase (Chd), the gene chd was first integrated into the chromosome of Bacillus subtilis WB800. High generation stability was achieved by almost no gene lost after six generations but Chd activity decreased. aprE promoter alteration, translation initiation region modification and multi-copy chromosome integration were studied and these modifications could increase Chd activity by 270%, 2304% and 25%. Chlorothalonil residual exhibited inhibition on bioconversion of lignocellulosic biomass. The addition of Chd crude enzyme (60 μL per g wheat straw) could increase glucose production by 36.10% and 39.65% in synergistic hydrolysis and separate hydrolysis by laccase and cellulase with 120 mg/L residual chlorothalonil. Filter paper activity and carboxymethyl cellulase activity were enhanced by 12.84% and 23.95%, and biomass of Trichoderma reesei was increased by 76.67% under 50 μg chlorothalonil/g dry straw in solid-state fermentation. Thus, the high-level stable expressed Chd effectively eliminated chlorothalonil inhibition on enzymatic hydrolysis and solid-state fermentation. It showed promising potential for bioremediation of chlorothalonil pollution and improving conversion efficiency of lignocellulose. [ABSTRACT FROM AUTHOR]

Abstract: The production of high titer xylanase without cellulase is required for prebleaching of pulps in pulp and paper industry. The mutant IITD3A of Melanocarpus albomyces developed from the spores of the wild type organism was used in this study. The statistical optimization of the process parameters by response surface methodology revealed that the production of xylanase was most affected by changes in the pH of the production medium which contained a soluble extract of wheat straw as the sole carbon source. When the pH of the production medium in a 14L bioreactor was controlled on-line at 7.8, xylanase activity of 415IUmL−1 was obtained after 36h fermentation. On cycling the pH between 7.8 and 8.2, the same activity could be attained in 24h with an overall productivity of 16,670IUL−1 h−1. The production of xylanase was also influenced by the fungus morphology; the activity being maximum when it exhibited pellet form at an agitation speed of 600rpm. On optimization of aeration rate to 0.25vvm, the xylanase activity further increased to 550IUmL−1 with a very high overall volumetric productivity of 22,000IUL−1 h−1. Thus, a 5.2-fold enhancement in overall volumetric productivity of xylanase could be obtained by the mutant in comparison to that obtained on insoluble wheat straw. [ABSTRACT FROM AUTHOR]

The stacking fermentation process plays a vital role in the production of sauce-flavour baijiu. The aim of this paper is to elucidate the effects of environmental variables on the fungal communities of different layers of fermented grains (zaopei) during the sixth round of stacking and the changes in volatile flavour substances during this process. The composition of the fungal communities in different layers during the stacking fermentation process was analysed. Principal coordinate analysis (pCoA) showed that the dominant fungal communities in different layers differed significantly with the stacking fermentation process. The dominant fungal genera were Thermoascus , Thermomyces and Issatchenkia. The total content of flavouring substances in the surface layer of zaopei were higher, but the types of flavouring substances were less than in the inner layer. The relationship between temperature, moisture content, acidity, starch content and reducing sugar content and microbial community was analysed by Redundancy analysis. The results showed that the correlation between microbial communities and physicochemical indexes in different layers of zaopei varied. The core fungal genera in the surface layer were mainly influenced by acidity, and the microorganisms in the inner layer were more strongly correlated with temperature. Spearman correlation coefficient revealed the correlation between fungal community and volatile flavour substances, and the results showed that microorganisms in different layers of zaopei have different correlations with flavour substances. This study contributes to the understanding of the evolution of different layers fungal communities during the stacking of sauce-flavour baijiu and their relationship with volatile aroma substances. [ABSTRACT FROM AUTHOR]

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100 rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0 at 40 °C. The scale-up was done based on the results of optimized parameters using 3 L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K 2 HPO 4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5–8.5, with thermal stability from 20 °C to 60 °C up to 180 min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4 °C and room temperature (35 °C) for 30 and 9 days respectively. The kinetic parameters K m (0.48 mg/ml) and V max (8.33 U/mg) were determined using birchwood xylan as substrate. • Use of rice bran and saw dust as substrates for xylanase production is possible. • Streptomyces geysiriensis yielded up to 93.97% xylanase in solid state fermentation. • Stability of xylanase was ranged from pH 6.5 to 8.5 and 20–60 °C up to 180 min. • Xylanase produced by S. geysiriensis was highly stable for 30 days at 4 °C. [ABSTRACT FROM AUTHOR]

Alkali-treated steam-exploded corn stover (SECSAT) was used as solid substrate for acetone–butanol–ethanol (ABE) production by absorbed lignocellulose fermentation (ALF) using Clostridium acetobutylicum ATCC 824. The ABE concentration in ALF culture had increased by 47% compared with that in submerged culture. More surprisingly, the acetone production was promoted and ethanol production was lower in the presence of SECSAT than that in its absence. ALF was also successfully in cofermentation of glucose and xylose, although decreased fermentability with an increase in the proportion of xylose. An invariable chemical composition and dry weight of SECSAT was found in ALF. Partial simultaneous saccharification and fermentation of SECSAT using a certain amount of cellulase could not only enhance the ABE concentration by 71%, but also significantly increase the area proportion of fiber cells in SECSAT from 53% to 90%, which would be an excellent paper making material. [Copyright &y& Elsevier]

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5–17.7 dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition. [Display omitted] [ABSTRACT FROM AUTHOR]

Due to the lack of available episomal plasmid, the improvement of many industrial strains, especially exogenous gene expression, is severely restricted. The failure of autonomous replication or low copy number of episomal plasmids is the main reason for the failure of many episomal plasmids construction. In this paper, Candida glycerinogenes , an industrial strain lacking episomal plasmids, was employed as the topic. A series of GFP-based plasmids containing autonomously replicating sequence (ARS) from different strain sources were constructed and analyzed for performance, and it was found that only the panARS from Kluyveromyces lactis compared with other nine low capacity ARSs proved to have the best performance and could be used to construct episomal plasmid. Further, the dual-ARS strategy was used to optimize the episomal plasmid, and the results indicated that only the dual-ARS plasmid +PPARS2 with double different ARSs, not the dual-ARS plasmid +panARS with double same ARSs, showed an improvement in all properties, with an increase in transformation efficiency of about 36% and a synchronous trend of fluorescence intensity and copy number, both by about 40%. In addition, constructed episomal plasmids were used to express the exogenous gene CrGES , and the fact that geraniol was found proved the versatility of the plasmids. The successful construction of episomal plasmids will also substantially facilitate genetic engineering research and industrial use of C. glycerinogenes in the future, as well as providing a feasible approach to create episomal plasmids for industrial strains. [ABSTRACT FROM AUTHOR]

The intracellular NAD(P)H insufficiency is the key factor which limits the reduced product (such as chiral alcohols) synthesis by whole cell biocatalysis or microbial cell factory. In this paper, we reported a novel solution to increase NADPH supply through strengthening the pentose phosphate pathway (PPP) flux with overexpression of extra zwf (gene for glucose 6-phosphatedehydrogenase) and glk (gene for glucokinase) by recombinant Escherichia coli BL21(DE3)/pETDuet-1- glk - zwf and pET28a- bccr containing a carbonyl reductase gene bccr. The amount of intracellular NADPH was significantly increased from 150.3 μmol/L to 681.8 μmol/L after strengthening the PPP flux, which was 4.5-fold to that of the control. It was applied to improve the asymmetric reduction of 4-chloroacetoacetate to ethyl S -4-chloro-3-hydroxybutylate catalyzed by the Bc CR, which increased the reaction yield 2.8-fold to the control. This strategy provides a new way to increase NADPH supply in E. coli cell factories. • A strategy was developed to enhance intracellular NADPH supply through increasing PPP flux. • Extra glk and zwf overexpression can increase PPP flux. • Intracellular NADPH increased from 150.3 μmol/L to 681.8 μmol/L more than 4.5-fold. • Yield of the asymmetric reduction of 4-chloroacetoacetate increased 2.8-fold. [ABSTRACT FROM AUTHOR]

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn2+ to Mn3+, a diffusible and strong non-specific oxidant capable of attacking bulky phenolic substrates. Therefore, MnP is indispensable in the polymer and paper industries. Previous attempts of MnP expression in Escherichia coli resulted in the formation of inclusion bodies which required in vitro refolding. Aiming to investigate the bacterial production of MnP, we have revealed an interesting mechanism underlying chaperone-assisted maturation of this enzyme to its active form. Since we previously found that in vitro expression of MnP in E. coli system depends on disulfide bond isomerase DsbC, we chose SHuffle T7 Express, an E. coli constitutively expressing DsbC, as the host for in vivo expression of MnP. Initially, only a low amount of the enzyme was present in the soluble fraction, with no detectable peroxidase activity. Co-expression of MnP with different chaperone revealed that DnaK, DnaJ, and GrpE contributed the most to the solubility improvement, however, remained in a complex with the MnP, preventing the enzyme to assume its active conformation. We resolved this by in vitro maturation, involving incubation of the MnP-chaperone complex with hemin, ATP, and ATP regeneration system. While ATP enables the chaperones to finish the refolding cycle and release the MnP in its correctly folded form, hemin supports the formation of the holo-enzyme with fully recovered peroxidase activity. We believe that the findings of this paper will serve as an important clue for establishing the bacterial production of fungal peroxidases in the future. Image 1 [ABSTRACT FROM AUTHOR]

In conventional forensic science, blood and urine have been used for drug testing. However, hair has recently attracted attention as a new source of biological information in this milieu. Drugs and biomolecules taken up by the hair from the capillaries of the scalp are retained in the hair without being degraded by enzymes, migrating toward the tip of the hair as the hair grows at a constant rate. As a result, drug residues are stored in the hair in chronological order. In recent years, mass spectrometry imaging (MSI) has been developed to visualize the history of drug use in hair samples, making use of this unique property. Advances in this drug testing technique are expected to create a powerful deterrent for drug abuse and doping. In this paper, we introduce the history of hair research using MSI and the evolution of instruments, matrices, and methods. [Display omitted] [ABSTRACT FROM AUTHOR]

The liquid phase was proved to be a significant influencing factor among the three phases in solid-state fermentation (SSF), which determined water control was crucial. However, obvious water loss was caused by microbial utilization and moisture evaporation. Super absorbent polymer (SAP) was utilized to supply water in SSF owing to its high water-holding capacity. Adding 0.15% SAP could significantly increase the biomass of Ganoderma lucidum by 33.59% and promote filter paper activity (FPA), endocellulase activity and laccase activity by 27.11%, 29.14% and 47.39%, respectively. Water states of fermentation substrates were detected by the low-field nuclear magnetic resonance (LF-NMR). Results revealed that water present and lost was dominated by the capillary water. At the end of fermentation, the capillary water content (C cw) in water-supply SSF was 20.48% and 17.20% higher than that in static SSF and cold-model SSF. The relaxation time of the capillary water was reduced by 56.53% in water-supply SSF and by 53.40% in static SSF, but it just reduced by 6.82% in cold-model SSF. In addition, the C cw in SSF had a high correlation with the biomass and lignocellulose-degrading enzyme activities of G. lucidum. These results clearly demonstrated that capillary water played a very important role in improved production of G. lucidum. • Liquid phase played a leading role in the three phases in SSF. • Water supply could promote growth and enzymes production of Ganoderma lucidum. • Water supply by 0.15% SAP increased biomass largest. • Water supply slowed down the increasing force between capillary water and substrate. • Capillary water content had a high correlation with biomass and enzymes activities. [ABSTRACT FROM AUTHOR]

Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-II Gt , previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-II Gt , produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-II Gt. Recombinant Cat-II Gt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529 μmol min−1 mg−1. The apparent K m and k cat values were 75 mM and 1.5 × 104 s−1 subunit−1, respectively. Recombinant Cat-II Gt was highly thermostable with a half-life of 30 min at 100°C. The structural attributes of Cat-II Gt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-II Gt a potential candidate for textile and paper processing industries. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate the medicinal mushroom Inonotus obliquus on the production of polysaccharides and changes of extracellular lignocellulolytic enzymes during submerged fermentation using alkali-treated birch sawdust as substrate. Meanwhile, in order to explore the degradation mode of lignocellulose in alkali-treated birch sawdust, degradation analysis of three components of lignocellulose was carried out. The fungus process in alkali-treated birch sawdust medium resulted in a higher degradation rate of cellulose, hemicellulose, and lignin of 39.24%, 51.00% and 31.3% after 11 days of submerged fermentation by the mycelium of I. obliquus , respectively. Maximal polysaccharide production and α-glucosidase inhibition rate determined in the alkali-treated birch sawdust medium were 6.93 mg/mL and 55.80%, while they were 4.98 mg/mL and 27.89% in the control. Moreover, high activities of laccase (51.95 IU/mL), CMCase (1.35 IU/mL), filter paper activity (0.50 IU/mL) and β-glucosidase (0.55 IU/mL) were observed in alkali-treated birch sawdust medium, respectively. The results demonstrated that the addition of alkali-treated birch sawdust could promote the yield and α-glucosidase inhibition activity of polysaccharides and induce the production of cellulase and xylanase, indicating that alkali pretreatment was conducive to utilization of birch sawdust by I. obliquus. [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens is a ubiquitous soil and plant-associated bacterial species which shows structural and adaptative responses to the environment. This present paper explores the ability of the strain L-17 to form subaerial biofilms on a liquid surface. Hydrophobic and non-wetting properties were observed for the rough top biofilm layer in contact with the air, which are quite different to the hydrophilic properties which were observed for the smooth biofilm layer in contact with the liquid. Both pellicle interfaces were visualized by scanning electron microscopy revealing a complex three-dimensional architecture composed of exopolymers organized in stacked fibrous network or sheet-like structures in the vicinity of the subaerial surface. Disruption of the extracellular matrix by combining physical and chemical treatments indicated that both loosely and tightly bound polysaccharides were found as major components of this complex pellicle. Proteins were also involved in the aggregation and cohesion of the matrix as multi extraction steps were needed to recover some tightly bounded proteins. This was confirmed by applying protease treatment which was able to significantly disrupt the pellicle. Overall results underline the ability of B. amyloliquefaciens L-17 to survive on air-liquid interfaces. This feature offers an interesting strategy to escape aquatic environments and develop aerial biofilm in response to environmental changes involving wet–dry cycles. [Display omitted] • Bacillus amyloliquefaciens pellicle exhibits differential physical properties between the air and the liquid interfaces. • Pellicle exhibits rough and hydrophobic features on the air interface with highly structured architecture. • Polysaccharides including cellulose-like polymers are the major components of the extracellular matrix. • Tightly bound proteins are also involved in the cohesion of the extracellular matrix. [ABSTRACT FROM AUTHOR]

Microorganisms are widely used to produce valuable compounds. Because thousands of metabolic reactions occur simultaneously and many metabolic reactions are related to target production and cell growth, the development of a rational design method for metabolic pathway modification to optimize target production is needed. In this paper, recent advances in metabolic engineering are reviewed, specifically considering computational pathway modification design and experimental evaluation of metabolic fluxes by 13C-metabolic flux analysis. Computational tools for seeking effective gene deletion targets and recruiting heterologous genes are described in flux balance analysis approaches. A kinetic model and adaptive laboratory evolution were applied to identify and eliminate the rate-limiting step in metabolic pathways. Data science-based approaches for process monitoring and control are described to maximize the performance of engineered cells in bioreactors. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer. [ABSTRACT FROM AUTHOR]

Photosynthetic bacteria (PSB) wastewater treatment is a novel technology for wastewater purification and resources recovery but is restricted by low efficiency. This paper applied zero-valent iron nanoparticles (Fe0 NPs) to enhance its performance. Results showed that 20 mg/L Fe0 NPs under light-anaerobic condition significantly increased the PSB biomass production and wastewater chemical oxygen demand removal by 122% and 164.3%, and shortened the time required for wastewater purification by 33%; these effects were far more better than the addition of Fe2+. The mechanism was because the addition of Fe0 NPs promoted the intracellular ATP content and pigments (carotenoid and bacteriochlorophyll) contents, and up-regulated dehydrogenase and succinate dehydrogenase activity; the increase rate reached 38.7%, 39.6%, 22.0%, 23.9% and 218.2%, respectively. Image 1 • Effects of Fe0 NPs on PSB was reported for the first time. • The COD removal was improved by 164.3% with 20 mg/L Fe0 NPs. • The biomass production was improved by 122% with 20 mg/L Fe0 NPs. • The mechanism of Fe0 NPs promotion was investigated and analyzed in detail. [ABSTRACT FROM AUTHOR]

Lignosulfonate is a by-product of the cooking process by sulfite pulping for paper manufacturing. The treatment of wood chips by various salts of sulfurous acid solubilizes lignin to produce a cellulose-rich wood pulp. Developing a technique for the conversion of lignosulfonate by-product to high value materials has an important industrial utility. Sphingobium sp. strain SYK-6, which was isolated from pulping wastewater, is one of the best enzymatically or genetically characterized bacteria for degrading lignin-derived aromatics. We have previously established a system for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), a novel platform chemical that can produce a variety of bio-based polymers, by introducing of ligA , ligB , and ligC genes from SYK-6 into a mutant strain of Pseudomonas putida PpY1100. In this study, extracts from lignosulfonates, which were desulphonated and depolymerized by alkaline oxidation, were evaluated as substrates for microbiological conversion to PDC by the transgenic bacteria. [ABSTRACT FROM AUTHOR]

Industrial scale microalgal cell disruption requires low cost, high efficiency and structural conservation of biomolecules for biorefinery. Many cultivated microalgae have thick walls and these walls are barriers for efficient cell disruption. Until recently, despite the high biodiversity of microalgae, little attention has been paid to thin-wall microalgal species in the natural environment for the production and recovery of valuable biomolecules. Instead of developing high power cell disruption devices, utilization of thin-wall species would be a better approach. The present paper describes a simple device that was assembled to evaluate the viability and effectiveness of biomolecule extraction from both thin- and thick-wall species as a proof of concept. This device was tested with high-pressure gases including N 2 , CO 2 plus N 2 , and air as the disruption force. The highest nitrogen pressure, 110 bar, was not able to disrupt the thick-wall microalgal cells. On the other hand, the thin-wall species was disrupted to different degrees using different pressures and treatment durations. In the same treatment duration, higher nitrogen pressure gave better cell disruption efficiency than the lower pressure. However, in the same pressure, longer treatment duration did not give better efficiency than the shorter duration. High pressure CO 2 treatments resulted in low soluble protein levels in the media. The best conditions to disrupt the thin-wall microalgal cells were 110 bar N 2 or air for 1 min among these tests. In these conditions, not only were the disruption efficiencies high, but also the biomolecules were well preserved. • The high biodiversity of microalgae implies many thin-wall species exist. • A device was assembled to break thin- and thick-wall algal cells using high pressure gases. • The efficiency of breaking the thin-wall cells was 100% using 110 bar N 2 or air but not for the thick-wall cells. • Cell wall thickness and thermo-tolerance are critical in microalgal screening. [ABSTRACT FROM AUTHOR]

Xylanases have useful applications in a wide range of industries. In this regard, Pichia pastoris has become one of the most attractive host platforms for large-scale production of xylanases. However, genomic engineering is still required for overexpression and efficient secretion. In this paper, we applied droplet-based method to screen directed evolved extracellular xylanase producing P. pastoris strain. Xylanase-producing P. pastoris cells were encapsulated in gel microdroplets with a fluorogenic reporter substrate. Improved production of xylanase increases fluorescence intensity of gel microdroplets, enabled accurate selection of evolved clones by droplet sorting. The screening strategy was validated by identifying yeast with improved xylanase production from a mixed sample with a positive selection accuracy of up to 98%. After three rounds of mutagenesis and selection, approximately 108 variants were screened, and a P. pastoris clone with more than 1.3-fold increase in xylanase activity was identified, representing cellular functions improvement of the production host. The throughput of this approach was at least 103-fold higher than that of the robot-assisted microtiter plate reader, and reagent consumption was reduced by ∼106-fold. Furthermore, the greatly shortened incubation time prior screening significantly accelerated the process of directed evolution. [ABSTRACT FROM AUTHOR]

In this paper, a biocompatible, non-toxic porous chitosan microcarrier was prepared by high voltage electrostatic field and freeze drying technology. The chitosan solution was pushed from the syringe drop into the sodium polyphosphate solution using a booster pump. The droplet diameter of the chitosan solution was adjusted by the voltage of the electrostatic field formed between the syringe and the sodium polyphosphate solution. The droplets were dropped into a sodium polyphosphate solution to form microspheres. The microspheres were subsequently immersed in 25% (v/v) glutaraldehyde for crosslinking to enhance the mechanical strength of the microspheres. These microspheres were then frozen and lyophilized to form a microcarrier. By performance characterization, these microcarriers had a particle size of 400–500 μm, a pore size of 15–20 μm, and a porosity of 90%. Under simulated human environmental conditions, the 21-day degradation rate was about 30%, indicating that the microcarriers have potential clinical value. [ABSTRACT FROM AUTHOR]

Biodiesel production depends to a great extent on the use of cheap raw materials, since biodiesel itself is a mass product, not a high-value product. New processing methods, such as micro-flow continuous processing combined with enzymatic catalysis, open doors to the latter. As reported here, the window of opportunity in enzyme-catalyzed biodiesel production is the conversion of waste cooking oil. The main technological challenge for this is to obtain efficient immobilization of the lipase catalyst on beads. The beads can be filled into tubular reactors where designed packed-bed provide porous channels, forming micro-flow. It turns out, that in this way, the immobilization costs become the decisive economic factor. This paper reports a solution to that issue. The use of oil cake enables economic viability, which is not given by any of the commercial polymeric substrates used so far for enzyme immobilization. The costs of immobilization are mirrored in the earnings and cash flow of the new biotechnological process. • Adding value and minimizing waste from the vegetable oil production. • Use of waste/by-product from oil industry in biodiesel production. • Economic feasibility of biodiesel production with immobilized lipase onto oil cake. • Biodiesel production in packed-bed micro-flow reactors with enzymes at industrial level. [ABSTRACT FROM AUTHOR]

Thermobifida alba AHK119 exhibits sufficient filter paper-degradation activity in its culture supernatant. AHK119-bMs (1365 bp) and AHK119-E5 (1425 bp), which encode novel GH5 family endoglucanases, were cloned from the genomic DNA of T. alba AHK119. AHK119-bMs and AHK119-E5 consisted of 454 and 474 amino acid residues, respectively, in which the catalytic domain (CD) and carbohydrate-binding module (CBM) were connected by an accessary module (linker region). The amino acid sequences of CD and CBM of AHK119-bMs were most identical to those of endo-β-mannanases (Man5As) from T hermobifida fusca TM51, T. halotolerans YIM90462, and T. cellulosilytica TB100. In contrast, the amino acid sequences of CD and CBM of AHK119-E5 were most identical to those of endo-1,4-β-glucanases (cellulases; Cel5As) from T. fusca and T. halotolerans YIM90462. However, the linker region of both the genes shared low identities with those of Man5As and Cel5As. AHK119-bMs showed broader specificities toward cellulosic substrates than Man5As, whereas AHK119-E5 showed higher activity toward insoluble cellulosic substrates than toward soluble ones, which was conflicting when compared with other Cel5As. In addition, AHK119-bMs and AHK119-E5 showed different requirements for metal ions from those of Man5As and Cel5As, respectively. Therefore, both the enzymes were identified as novel GH5 endoglucanases, and the accessary modules seemed to play important roles in their enzymatic properties. [ABSTRACT FROM AUTHOR]

Nowadays, the pretreatment of wastewater prior to discharge is very important in various industries as the wastewater without any treatment contains high organic pollution loads that would pollute the receiving waterbody and potentially cause eutrophication and oxygen depletion to aquatic life. The reuse of seafood wastewater discharge in microalgae cultivation offers beneficial purposes such as reduced processing cost for wastewater treatment, replenishing ground water basin as well as financial savings for microalgae cultivation. In this paper, the cultivation of Chlorella vulgaris with an initial concentration of 0.01 ± 0.001 g⋅L−1 using seafood sewage discharge under sunlight and fluorescent illumination was investigated in laboratory-scale without adjusting mineral nutrients and pH. The ability of nutrient removal under different lighting conditions, the metabolism of C. vulgaris and new medium as well as the occurrence of auto-flocculation of microalgae biomass were evaluated for 14 days. The results showed that different illumination sources did not influence the microalgae growth, chemical oxygen demand (COD) and biochemical oxygen demand (BOD) significantly. However, the total nitrogen (total-N) and total phosphorus (total-P) contents of microalgae were sensitive to the illumination mode. The amount of COD, BOD, total-N and total-P were decreased by 88%, 81%, 95%, and 83% under sunlight mode and 81%, 74%, 79%, and 72% under fluorescent illumination, respectively. Furthermore, microalgae were auto-flocculated at the final days of cultivation with maximum biomass concentration of 0.49 ± 0.01 g⋅L−1, and the pH value had increased to pH 9.8 ± 0.1 under sunlight illumination. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

An inducer is crucial for cellulase production. In this study, duckweed was used as an inducer of cellulase production by Trichoderma reesei RUT C30. In a reaction induced by 50 g/L duckweed in shake flasks, the filter-paper activity (FPA) reached 6.5 FPU/mL, a value comparable to that induced by avicel. The enzyme-hydrolysis rate induced by steam-exploded corn stalk was 54.2%, representing a 28% improvement over that induced by avicel. The duckweed starch was hydrolyzed to glucose, which was subsequently used for biomass accumulation during the fermentation process. Furthermore, to optimize the control of the fermentation process, a combined substrate of avicel and duckweed was used to induce cellulase production by T. reesei RUT C30. The cellulase production and hydrolysis rates of the combined substrate, compared with avicel alone, were 39.6% and 36.7% higher, respectively. The results of this study suggest that duckweed is a good inducer of cellulase production in T. reesei , and it might aid in decreasing the cost of lignocellulosic materials hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract: The adaptation of inkjet printing technology for the realisation of controlled micro- and nano-scaled biological structures is of great potential in tissue and biomaterial engineering. In this paper we present the Olivetti BioJet system and its applications in tissue engineering and cell printing. BioJet, which employs a thermal inkjet cartridge, was used to print biomolecules and living cells. It is well known that high stresses and forces are developed during the inkjet printing process. When printing living particles (i.e., cell suspensions) the mechanical loading profile can dramatically damage the processed cells. Therefore computational models were developed to predict the velocity profile and the mechanical load acting on a droplet during the printing process. The model was used to investigate the role of the stiffness of the deposition substrate during droplet impact and compared with experimental investigations on cell viability after printing on different materials. The computational model and the experimental results confirm that impact forces are highly dependent on the deposition substrate and that soft and viscous surfaces can reduce the forces acting on the droplet, preventing cell damage. These results have high relevance for cell bioprinting; substrates should be designed to have a good compromise between substrate stiffness to conserve spatial patterning without droplet coalescence but soft enough to absorb the kinetic energy of droplets in order to maintain cell viability. [Copyright &y& Elsevier]

β-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude β-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that β-galactosidase from L. acidophilus shows a particular specificity towards the formation of β-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C). Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Lignin is an aromatic polymer that makes a network by intertwining between cellulose fibers in plant. As the lignin network retards access to carbohydrates, it is regarded as a nuisance during biomass processing. When wood is processed into paper pulp or bioethanol, lignin is produced as a by-product and utilized as fuel or a soil amendment. Recently, there has been much interest in the aromatic structure of lignin in relation to the utilization of lignocellulose and the search for petroleum substitutes. Sulfur-free pulping methods, such as soda-anthraquinone cooking, provide more opportunity for using lignin than the alternative kraft process. Our aim was to expand the availability of soda lignin from Japanese cedar, the most planted tree in Japan, by fungal degradation. We performed degradation assays to identify suitable fungi for the efficient breakdown of soda lignin from cedar. Fourteen fungi from both white-rot and leaf-litter fungi were identified using the RBBR and Sundman and Näse assays. By nuclear magnetic resonance analysis we obtained water- and/or methanol-soluble degradation products from four fungi, and the patterns indicate specific degradation mechanisms for each fungi. These results suggest that the screened fungi have more than one mechanism for degrading soda lignin from Japanese cedar. [ABSTRACT FROM AUTHOR]

Development of industrially potent cellulolytic enzymes is one of the greatest challenges faced in lignocellulosic feed-stock based bio-refining. In the current work cross-linked enzyme aggregates (CLEAs) of commercial cellulase mix were successfully prepared and their performance to be used as potential industrial enzymes in terms of stability and wheat straw hydrolysis was evaluated. The CLEAs were more stable compared to native enzymes with half-lives being 2.30-, 1.56-, 3.07- and 1.67-fold higher at 70°C for filter paper activity (FPA), endoglucanase, β-glucosidase and xylanase, respectively. CLEAs retained 77.4% of endoglucanase and 85.9% of xylanase activity after five cycles of hydrolysis of soluble substrates such as carboxymethyl cellulose and xylan, respectively. A maximum saccharification yield of 31.8% by soluble enzymes and 32.9% by CLEAs were obtained when alkali-pretreated wheat straw was subjected to hydrolysis. On repeated batch hydrolysis for five consecutive cycles of 24 h each, the CLEAs showed an overall higher saccharification yield of 43.3% compared to 31.8% with soluble enzymes. Highlights • Cross-linked enzyme aggregates of cellulolytic enzymes were prepared. • The kinetic and stability parameters of the best CLEA were determined. • The best CLEA was more effective in hydrolysing wheat straw. • It resulted in overall saccharification yield of 43.3% compared to 31.8% with soluble enzymes. [ABSTRACT FROM AUTHOR]

Abstract: A WHO position paper states that allergen immunotherapy is an effective treatment for allergic diseases, and well characterized allergens should be used in immunotherapy. The house dust mite is a major cause of allergic disease. However, the biological activity of the mite extracts currently used cannot be clearly determined, since these extracts contain various impurities. The use of recombinant allergens can avoid this problem. However, there remains a risk of contamination by other impurities, such as host cell-derived proteins (HCPs). Advanced purification techniques are thus required to remove these contaminants. C8/119S is a mutant of the major house dust mite allergen Der f 2, and is expressed and accumulated as an inclusion body in Escherichia coli. The C8/119S was refolded and purified through three column chromatography steps. Using this method, we could obtain about 2g of the purified C8/119S in one purification batch. This amount is equivalent to 100,000 of the maintenance doses required for immunotherapy based on the WHO position paper. The purity of the C8/119S was 99% or more. The antigenicity of HCPs in the C8/119S was examined by passive cutaneous anaphylaxis assays. When the C8/119S was administered at 40μg/kg, no local anaphylaxis was observed. C8/119S was thus highly purified with an extremely low level of impurities, and our procedure was shown to be an effective advanced production-scale purification process for this Der f 2 mutant. In this study, we established an advanced purification processes for C8/119S, then characterized the purified C8/119S and evaluated its purity. [ABSTRACT FROM AUTHOR]

Magnetite cationic liposomes (MCLs) have a positive surface charge and are used as a heating mediator for hyperthermia, because they generate heat in an alternating magnetic field (AMF) due to hysteresis loss. In our previous paper, hyperthermia using MCLs was applied to animals having several types of tumors in mice, rats, hamsters, and rabbits, and a strong anticancer effect was observed. For mice, complete tumor regression was observed when the tumor size was smaller than 5 mm. However, a protocol for large tumors is necessary for the clinical application. In the present paper, a protocol for tumors with a size greater than 15 mm in mice was investigated. MCLs were injected into an MM46 tumor (size, 15 mm) in C3H/HeN mice, which were subjected to AMF for 30 min. The temperature at the surface of the tumor reached 45°C and was maintained by controlling the magnetic field intensity. Hyperthermia treatment was repeated twice with 24-h intervals (repeated hyperthermia; RH), and RH was carried out until complete tumor regression was observed. Complete tumor regression was achieved in all mice treated once, twice or six times with RH, and mice acquired antitumor immunity. This protocol, which is termed frequent RH, is a potent approach for cancer therapy. [Copyright &y& Elsevier]

Polyhydroxyalkanoates (PHA) are naturally occurring biopolymers, obtained from microorganisms. Properties like biodegradability and biocompatibility make PHA a part of today's commercial polymer industry. However, the production cost of PHA has been a great barrier to extend its application to large scale production. Substrates and usage of pure cultures constitute the main reason for its high production cost. On the other hand, rapid industrialization i.e., industrial sectors such as sugar, pulp and paper, fruit and food processing, dairies, slaughterhouses, and poultries, has resulted in the generation of the huge quantity of wastes. Consequently, becoming large source of environmental pollution and health hazard. This review emphasizes on the usage of various waste feedstocks obtained from industrial and agricultural industries as an alternate substrate for PHA production. As these waste materials are rich in organic material and also microbes, they can be the good starting material for PHA production. Additionally, advantages and economic importance of mixed cultures and also PHA applications are discussed. Future prospects and challenges in PHA production from waste feedstocks are also highlighted. [ABSTRACT FROM AUTHOR]

Xylanase is an important enzyme involved in degrading xylan. In this study, an extracellular cellulase-free, thermostable endo-xylanase which was produced by Streptomyces griseorubens LH-3 with bagasse semi-cellulose as a carbon source was purified and characterized. The xylanase was purified 4-fold with a recovery yield of 21.6% by precipitation with 25–55% (NH 4 ) 2 SO 4 , Mono Q ion exchange chromatography and sephacryl S-200 HR gel filtration chromatography. It appeared as a monomeric protein on SDS-PAGE gel and had an apparent molecular weight of 45.5 kDa with specific activity of 434 IU/mg. Using birchwood xylan as substrate, the maximum velocity ( V max ) and Michaelis–Menten constant ( K m ) were found to be 1.44 mg/ml and 2.05 μmol/min mg, respectively. The purified xylanase was active at pH 4.0–8.0 with an optimum pH of 5.0. It was stable at temperatures between 30°C and 50°C, exhibiting maximum activity at 60°C. Hg 2+ and Al 3+ inhibited the enzyme activity significantly. Enzymatic product analysis indicated that the enzyme was an endo-xylanase, whose hydrolysis products were mainly a series of short-chain xylooligosaccharides. Furthermore, it was used for biobleaching of eucalyptus kraft pulp, and results showed that this purified xylanase increased the brightness of the pulp by 14.5% and reduced the kappa number by 24.5%. All these industrially relevant characteristics made it had potential application in the pulp and paper industry as a biobleaching agent. [ABSTRACT FROM AUTHOR]

Illigera henryi , an endemic traditional Chinese medicine, contains abundant aporphine alkaloids that possess various bioactivities. In the present study, tubers of I. henryi were fermented by several fungi, and the acetylcholinesterase (AChE) inhibitory activities of non-fermented and fermented I. henryi were measured. The results showed that the fermentation of I. henryi with Clonostachys rogersoniana 828H2 is effective for improving the AChE inhibitory activity. A key biotransformation was found during the C. rogersoniana fermentation for clarifying the improvement of the AChE inhibitory activity of I. henryi : ( S )-actinodaphnine ( 1 ) was converted to a new 4-hydroxyaporphine alkaloid (4 R ,6a S )-4-hydroxyactinodaphnine ( 2 ) that possessed a stronger AChE inhibitory activity, with an IC 50 value of 17.66 ± 0.06 μM. This paper is the first to report that the pure strain fermentation processing of I. henryi and indicated C. rogersoniana fermentation might be a potential processing method for I. henryi . [ABSTRACT FROM AUTHOR]

Hydroxylation of steroids at various positions is a powerful tool for the production of valuable pharmaceutical ingredients and precursors. Our paper reported the synchronous dihydroxylation of an efficient strain, i.e., Colletotrichum lini AS3.4486, at two points. C. lini AS3.4486 was selected from 10 strains; this strain can catalyze the dihydroxylation of androst-1,4-dien-3,17-dione at C-11α and C-15α positions. Transformation of ADD(I) by C. lini AS3.4486 produced metabolites II–IV. The structures of these compounds were elucidated by liquid chromatography–mass spectrometry (LC–MS), Fourier Transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and X-ray as 15-hydroxyandrost-1,4-dien-3,17-dione (15α-OH-ADD; II), 11,15-dihydroxyandrost-1,4-dien-3,17-dione (11,15-diOH-ADD; III), and 15,17β-dihy-droxyandrost-1,4-dien-3-one (15-OH-BD; BD is the abbreviation of boldenone; IV). III, as a novel compound, was reported for the first time. The course of conversion and mechanism about dihydroxylation reaction was also investigated. On the basis of time course analysis of hydroxylation, I underwent regioselective hydroxylation at 15 position and was subsequently converted to III and IV. Enzyme inhibition analysis showed that 11- and 15-hydroxylations were catalyzed by different hydroxylases. The effect of substrate concentration on I transformation was also determined. Results showed that the optimum concentration of I was 20 g/L, and the yield of III was up to 18.8 g/L. [ABSTRACT FROM AUTHOR]

Recently, the demand for d -amino acid profiling has been drastically increasing because the significance of d -amino acid in various biological events is suggested. However, the present methodologies for d -amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for d -amino acid profiling must be developed. In this paper, we developed a novel method for d -amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for d -amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the d -amino acid research in the future. [ABSTRACT FROM AUTHOR]

This paper explores the use of low-frequency–low-amplitude acoustic vibration on biofilm formation. Biofilm development is thought to be governed by a diverse range of environmental signals and much effort has gone into researching the effects of environmental factors including; nutrient availability, pH and temperature on the growth of biofilms. Many biofilm-forming organisms have evolved to thrive in mechanically challenging environments, for example soil yet, the effects of the physical environment on biofilm formation has been largely ignored. Exposure of Pseudomonas aeruginosa to vibration at 100, 800 and 1600 Hz for 48 h, resulted in a significant increase in biofilm formation compared with the control, with the greatest growth seen at 800 Hz vibration. The results also show that this increase in biofilm formation is accompanied with an increase in P. aeruginosa cell number. Acoustic vibration was also found to regulate the spatial distribution of biofilm formation in a frequency-dependent manner. Exposure of Staphylococcus aureus to acoustic vibration also resulted in enhanced biofilm formation with the greatest level of biofilm being formed following 48 h exposure at 1600 Hz. These results show that acoustic vibration can be used to control biofilm formation and therefore presents a novel and potentially cost effective means to manipulate the development and yield of biofilms in a range of important industrial and medical processes. [ABSTRACT FROM AUTHOR]

PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB − 4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated. [ABSTRACT FROM AUTHOR]

Microcapsule entrapped low density cells with culture (ELDC wc ), different from free cell culture, conferred stronger stress resistance and improved cell viability of microorganisms. In this paper, the quorum sensing (QS) system of Vibrio harveyi was used to investigate changes when cells were cultured in microcapsules. Cells in ELDC wc group grew into cell aggregates, which facilitated cell–cell communication and led to increased bioluminescence intensity. Moreover, the luxS -AI-2 system, a well-studied QS signal pathway, was detected as both luxS gene and the AI-2 signaling molecule, and the results were analyzed with respect to QS capacity of unit cell. The V. harveyi of ELDC wc also showed higher relative gene expression and stronger quorum sensing capacity when compared with free cells. In conclusion, the confined microcapsule space can promote the cell aggregates formation, reduce cell–cell communication distance and increase local concentration of signal molecule, which are beneficial to bacterial QS. [ABSTRACT FROM AUTHOR]

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o -diphenols and the oxidation of o -diphenols into o -quinones that form brown or black pigments. In the present paper, cefotaxime, a cephalosporin antibacterial drug, was tested as an inhibitor of tyrosinase. The results show that cefotaxime inhibits both the monophenolase and diphenolase activities of tyrosinase. For the monophenolase activity, cefotaxime increased the lag time and decreased the steady-state activity with an IC 50 of 3.2 mM. For the diphenolase activity, the inhibition by cefotaxime is reversible and mix-I type with an IC 50 of 0.14 mM. The inhibition constants ( K I and K IS ) were determined to be 0.14 and 0.36 mM, respectively. The molecular mechanism of inhibition of tyrosinase by cefotaxime was determined by fluorescence quenching and molecular docking. The results demonstrated that cefotaxime was a static quencher of tyrosinase and that cefotaxime could dock favorably in the active site of tyrosinase. This research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors in the future. [ABSTRACT FROM AUTHOR]

The current paper has elaborated the efficient utilization of non-edible oil seed cakes (NEOC), by-products of the bio-diesel extraction process to develop a herbal and novel mosquitocidal composition against the Aedes aegypti larvae. The composition consisted of botanical active ingredients, inerts, burning agents and preservatives; where the botanical active ingredients were karanja ( Pongamia glabra ) cake powder and jatropha ( Jatropha curcas ) cake powder, products left after the extraction of oil from karanja and jatropha seed. The percentage mortality value recorded for the combination with concentration, karanja cake powder (20%) and jatropha cake powder (20%), 1:1 was 96%. The coil formulations developed from these biodiesel by-products are of low cost, environmentally friendly and are less toxic than the synthetic active ingredients. [ABSTRACT FROM AUTHOR]

Purification of cyclodextrin glycosyl transferase (CGTase) from Bacillus cereus using polyethylene glycol (PEG)-potassium phosphates aqueous two-phase flotation (ATPF) system was studied in this paper. The effects of varying PEG molecular weight, tie-line length (TLL) value, volume ratio (V R ), pH value, crude concentration and gas nitrogen flotation time were investigated. The optimal condition for purification of CGTase was attained at 18.0% (w/w) PEG 8000, 7.0% (w/w) potassium phosphates, V R of 3.0, 20% (w/w) crude load at pH 7, and 80 min nitrogen flotation time at a flow rate of 5 L/min. With this optimal condition, purification factor (P FT ) of 21.8 and a yield (Y T ) of 97.1% were attained. CGTase was successfully purified in a single downstream processing step using the ATPF. [ABSTRACT FROM AUTHOR]

In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length ( TLL 2 ). This relationship provides good fits ( r 2 > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0–50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement ( r 2 = 0.94) with the proposed relationship, ln K = α′ pH + β′ . These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load. [ABSTRACT FROM AUTHOR]