Huayou Chen

Effects of rhizobia inoculation and nitrogen fertilization on photosynthetic physiology of soybean

Plant growth, contents of photosynthetic pigments, photosynthetic gas exchange, and chlorophyll (Chl) fluorescence in soybean [Glycine max (L.) Merr. cv. Heinong37] were investigated after it was inoculated with Sinorhizobium fredii USDA191 or treated with 5 mM (NH4)2SO4 (N5) and 30 mM (NH4)2SO4 (N30), respectively. In the plants following N5 fertilization, not only plant biomass, leaf area, and Chl content, but also net photosynthetic rate (PN), stomatal conductance (gs), carboxylation efficiency (CE), maximum photochemical efficiency (Fv/Fm) of photosystem 2 (PS2), and quantum yield of PS2 (ΦPS2) were markedly improved as compared with the control plants. There were also positive effects on plant growth and plant photosynthesis after rhizobia inoculation, but the effects were much less than those of N5 fertilization. For N30 plants there were no significant positive effects on plant growth and photosynthetic capacity. Plant biomass, PN, and gs were similar to those of N-limited (control) plants. ΦPS2 and photochemical quenching (qP) were obviously declined while content of carotenoids and non-photochemical quenching (qN) were significantly enhanced in N30 treated plants. This indicated that excess N supply may cause some negative effects on soybean plants.

Surface display of the thermophilic lipase Tm1350 on the spore of Bacillus subtilis by the CotB anchor protein

Lipases expressed in microbial hosts have great commercial value, but their applications are restricted by the high costs of production and harsh conditions used in industrial processes, such as high temperature and alkaline environment. In this study, an Escherichia coli–Bacillus subtilis shuttle vector (pHS-cotB-Tm1350) was constructed for the spore surface display of the lipase Tm1350 from hyperthermophilic bacterium Thermotoga maritima MSB8. Successful display of the CotB-Tm1350 fusion protein on spore surface was confirmed by Western blot analysis and activity measurements. The optimal catalytic temperature and pH of the spore surface-displayed Tm1350 were 80xa0°C and 9, respectively, which were higher than non-immobilized Tm1350 (70xa0°C and pH 7.5). Analysis of thermal and pH stability showed that spore surface-displayed Tm1350 retained 81 or 70xa0% of its original activity after 8xa0h of incubation at pH 8 or pH 9 (70xa0°C), which were 18xa0% higher than the retained activity of the non-immobilized Tm1350 under the same conditions. Meanwhile, recycling experiments showed that the recombinant spores could be used for up to three reaction cycles without a significant decrease in the catalytic rate (84xa0%). These results suggested that enzyme display on the surface of the B. subtilis spore could serve as an effective approach for enzyme immobilization, which has potential applications in the harsh biochemical industry.

Structural and energetic insights into the selective interactions of monoacylglycerol lipase with its natural substrate and small-molecule inhibitors

The monoacylglycerol lipase (MAGL) regulates 2-arachidonoyl glycerol (2-AG) level in the endocannabinoid system (ECS), which is implicated in a number of severe diseases such as cancer and Alzheimer’s disease. However, most existing MAGL inhibitors also show additional inhibitory effects on fatty acid amide hydrolase (FAAH), another member of the ECS that degrades the 2-AG analog N-arachidonoyl ethanolamine. Understanding of molecular mechanism and biological implication underlying the specific interactions in MAGL–ligand recognition is thus fundamentally important for the rational design of selective MAGL inhibitors. In the current study, the structural basis and energetic property regarding the binding of several MAGL inhibitors as well as its substrate 2-AG to both the MAGL and FAAH are investigated systematically by integrating molecular docking, quantum mechanics/molecular mechanics analysis, and Poisson–Boltzmann/surface area solvent model. In addition, a novel quantitative structure–selectivity relationship method is proposed to help in the explanation and prediction of inhibitor selectivity between MAGL and FAAH. It is suggested that the selectivity is primarily determined by the size, topology, and property of the rear moiety of inhibitor compounds; a bulky, bifurcated rear is the prerequisite for a inhibitor to have high selectivity for MAGL over FAAH, whereas those dual-type MAGL–FAAH inhibitors should possess a small, rear moiety—the ideal choice is a single aromatic branch occupying this position.

Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis

Spore surface display is the most desirable with enhanced effects, low cost, less time consuming and the most promising technology for environmental, medical, and industrial development. Spores have various applications in industry due to their ability to survive in harsh industrial processes including heat resistance, alkaline tolerance, chemical tolerance, easy recovery, and reusability. Yeast and bacteria, including gram-positive and -negative, are the most frequently used organisms for the display of various proteins (eukaryotic and prokaryotic), but unlike spores, they can rupture easily due to nutritive properties, susceptibility to heat, pH, and chemicals. Hence, spores are the best choice to avoid these problems, and they have various applications over nonspore formers due to amenability for laboratory purposes. Various strains of Clostridium and Bacillus are spore formers, but the most suitable choice for display is Bacillus subtilis because, according to the WHO, it is safe to humans and considered as “GRAS” (generally recognized as safe). This review focuses on the application of spore surface display towards industries, vaccine development, the environment, and peptide library construction, with cell surface display for enhanced protein expression and high enzymatic activity. Different vectors, coat proteins, and statistical analyses can be used for linker selection to obtain greater expression and high activity of the displayed protein.

Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis

In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 mM dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.

Cations Optimization for Protein Enrichment in Rice Straw by Mixed Cultures of Neurospora crassa 14-8 and Candida utilis Using Response Surface Methodology

This study investigated the effect of cation optimization by mixed cultures of Neurospora crassa and Candida utilis on the true protein (TP) content. Firstly, to enhance the nutritional contents of rice straw (RS), two fermentation parameters (effect of inoculation ratio and inoculation time) were optimized. It was found that when C. utilis was inoculated 60xa0h later than N. crassa with the inoculation ratio of 1:5 (N. crassa to C. utilis), the maximum TP yield was obtained. In order to further optimize TP content, Plackett-Burman design (PBD) and Box-Behnken design (BBD) of response surface methodology (RSM) were adopted. The results of PBD indicated that Mn2+, Zn2+, and Cu2+ were the significant variables. The optimum values for the three cations determined by the BBD were as follows: Mn2+ 0.06xa0g/L, Zn2+ 0.15xa0g/L, and Cu2+ 0.2xa0g/L. After the optimization of RSM, a model was proposed to predict the optimum value 10.36% confirmed by the experimental result 9.84%. The TP content increased from 3.98 to 9.84%, with 147.24% of its increase rate. This study proposed an ecofriendly and economical way to convert RS into protein-enriched livestock feed.

Impact of orientation and flexibility of peptide linkers on T-maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)4] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS)4) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80xa0°C after 5xa0h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.

Protein Production Through Microbial Conversion of Rice Straw by Multi-Strain Fermentation

Multi-strain mixed fermentation can provide a relatively complete lignocellulosic enzyme system compared with single-strain fermentation. This study was firstly to screen strains which have a strong ability to hydrolyse rice straw (RS) enzymatically and enrich true protein (TP). Then, the conditions in the process of SSF, including the optimum inoculum size of mixed strains, inoculation ratio, and different inoculation time of N. crassa 14–8, were optimized. The experimental results showed that the highest TP content could be obtained by using N. crassa 14–8, C. utilis, and P. chrysosporium as mixed strains, and 5xa0mM Mn2+ and 50xa0mM veratryl alcohol were used as inducers of lignin peroxidase (LiP) to improve the efficiency of enzymatic hydrolysis. When N. crassa 14–8 was inoculated 1xa0day later than P. chrysosporium, the total inoculum size was 10%, and the optimum ratio of N. crassa 14–8 to P. chrysosporium was 1:2, the maximum TP yield (8.89%) was obtained, with 123.37% of its increase rate. This work proposed a technique with potential application in large-scale feedstuff protein conversion.