The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber

BACKGROUND: The conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming. RESULTS: The gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a K(m) of 0.09 mg/ml and a V(max) of 18.13 μmol/min. K(+), Li(+), Ni(2+) and Sr(2+) showed little or no inhibitory effect on PEL168P activity, and Ca(2+) enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization. CONCLUSIONS: Pectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming

Modellera effekten av klimatförändringar och socioekonomiska förändringar på näringsämnesdynamiken i Vombsjöns avrinningsområde

Climate change and socio-economic development are greatly affecting the quality of freshwater, especially the excessive accumulation of nutrients (N and P), which eventually leads to the occurrence of eutrophication. Lake Vomb is one of the main sources of drinking water in southern Sweden and the nutrient load from the catchment makes it a eutrophic lake with recurring algal blooms. This project developed a hydrological model by Soil and Water Assessment Tool (SWAT) to model the impact of climate and socio-economic changes on nutrient dynamics on Lake Vomb. Two combinations of future scenarios were selected as combinations of Representative Concentration Pathways (RCP) and Shared Socioeconomic Pathways (SSP): 1) SSP1/RCP4.5 and 2) SSP5/RCP8.5. For the calibration and validation period, satisfactory results were obtained for monthly flow (R2 and NSE were 0.92 and 0.82, respectively) and yearly nutrient load simulation obtained overall convincing results (R2, NSE and PBIAS were 0.67, 0, 17% for total nitrogen (TN) and 0.78, -0.11, -1% for total phosphorus (TP) at the main inflow and R2, NSE and PBIAS were 0.46, 0.27, 15% for TN and 0.62, -0.01, -0.06% for TP at the second inflow). Simulation results of increased future flow scenarios showed higher peaks under the RCP8.5 scenario than under RCP4.5. Also, there was an increasing trend that flow will continuously rise during the simulation period. Results from future nutrient load simulation showed that the TN load was below the baseline in most scenarios and the TP load was all above the baseline. Besides, nutrient loading is more sensitive to the combination of SSPs and RCPs and got the highest loads under RCP4.5/SSP1. The challenge of reducing nutrient load increases over time, especially for P because the simulated load was higher for 2090-2100 than the load for 2040-2050. This project has limitations such as uncertain agriculture activities input and nutrient observations. This project provides projections of flows and nutrient loads into Lake Vomb for a combination of possible future scenarios and more different scenarios such as different RCPs and different designed SSPs deserve to be studied in the future

Modellera effekten av klimatförändringar och socioekonomiska förändringar på näringsämnesdynamiken i Vombsjöns avrinningsområde

Climate change and socio-economic development are greatly affecting the quality of freshwater, especially the excessive accumulation of nutrients (N and P), which eventually leads to the occurrence of eutrophication. Lake Vomb is one of the main sources of drinking water in southern Sweden and the nutrient load from the catchment makes it a eutrophic lake with recurring algal blooms. This project developed a hydrological model by Soil and Water Assessment Tool (SWAT) to model the impact of climate and socio-economic changes on nutrient dynamics on Lake Vomb. Two combinations of future scenarios were selected as combinations of Representative Concentration Pathways (RCP) and Shared Socioeconomic Pathways (SSP): 1) SSP1/RCP4.5 and 2) SSP5/RCP8.5. For the calibration and validation period, satisfactory results were obtained for monthly flow (R2 and NSE were 0.92 and 0.82, respectively) and yearly nutrient load simulation obtained overall convincing results (R2, NSE and PBIAS were 0.67, 0, 17% for total nitrogen (TN) and 0.78, -0.11, -1% for total phosphorus (TP) at the main inflow and R2, NSE and PBIAS were 0.46, 0.27, 15% for TN and 0.62, -0.01, -0.06% for TP at the second inflow). Simulation results of increased future flow scenarios showed higher peaks under the RCP8.5 scenario than under RCP4.5. Also, there was an increasing trend that flow will continuously rise during the simulation period. Results from future nutrient load simulation showed that the TN load was below the baseline in most scenarios and the TP load was all above the baseline. Besides, nutrient loading is more sensitive to the combination of SSPs and RCPs and got the highest loads under RCP4.5/SSP1. The challenge of reducing nutrient load increases over time, especially for P because the simulated load was higher for 2090-2100 than the load for 2040-2050. This project has limitations such as uncertain agriculture activities input and nutrient observations. This project provides projections of flows and nutrient loads into Lake Vomb for a combination of possible future scenarios and more different scenarios such as different RCPs and different designed SSPs deserve to be studied in the future

Impact of different light sources on the responses of moth

In recent decades, the negative effects of artificial light at night on natural ecosystems have attracted the attention of ecologists. Studies have shown that artificial light at night leads to a considerable reduction in insects and has a worrying impact on terrestrial ecosystems, including nocturnal insects (e.g. Lepidoptera) such as moths. Warm white light is generally expected to have a lower ecological impact compared with cold white light which has a higher proportion of blue light (< 500 nm). The aim of this study is to investigate the impact of three light emitting diode (LED) light sources with different spectral power distributions on the responses of the greater wax moth (Galleria mellonella) under controlled experimental settings. In this experiment, three light sources with different spectral power distribution and dark condition were used and the start response time, time active, time flap wings, time flap wings / time active, main activity area and stop area of the greater wax moth were investigated. The light treatment used were: (1) darkness (n = 13); (2) warm-white light (correlated color temperature of 2675 K, n = 12); (3) white light (4070 K, n = 4); (4) cold white light (6200 K, n = 8). The experiment was performed in a rectangular light-tight box under controlled conditions. Main activity area showed significant difference between warm-white light and cold white light. With cold white light, a larger proportion of the moths were active in the area with the highest light levels, while the main activity area in the warm white light was in the zone with the lowest light levels. Other variables, however, did not show significant differences. The conclusion is that warm white light had a lower ecological impact than cold white light due to a larger proportion of moth is attracted to areas with stronger light. The results support the notion that in outdoor environments, warm white LEDs (maximum 3000 K) should be preferred compared to cold white light LEDs (over 3000 K), to reduce the impact on insects such as moths. The low correlated color temperature light sources used outdoors deserves more in-depth development and research

A multi-objective brain storm optimization for integrated distributed flexible job shop and distribution problems

Production and distribution are critical components of the furniture supply chain, and achieving optimal performance through their integration has become a vital focus for both the academic and business communities. Moreover, as economic globalization progresses, distributed manufacturing has become a pioneering production technique. Via leveraging a distributed flexible manufacturing system, mass flexible production at lower costs can be achieved. To this end, this study presents an integrated distributed flexible job shop and distribution problem to minimize makespan and total tardiness. In our research, a set of custom furniture orders from different customers are processed among flexible job shops and then delivered by vehicles to customers as the due date. To distinctly show the presented problem, a mixed integer mathematical programming model is created, and a multi-objective brain storm optimization method is introduced considering the problem's features. In comparison to the other three advanced methods, the superiority of the algorithm created is showcased. The findings of the experiments demonstrate that the constructed model and the introduced algorithm have remarkable competitiveness in addressing the problem being examined

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Abstract Background β-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. Results A thermo-alkaline β-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0–11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing β-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg−1 was observed on konjac glucomannan with the K m and k cat value of 0.62 g l−1 and 1238.9 s−1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml−1 with secretory rate of 98.5% was obtained using SP lipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml−1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. Conclusions The thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml−1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline β-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other β-mannanases in B. subtilis