Metagenomic and proteomic analysis of bacterial retting community and proteome profile in the degumming process of kenaf bast.

BACKGROUND: Data on the microbial community and functional proteins associated with degumming in kenaf remains scant. Here, we analyzed the microbial communities associated with kenaf (Hibiscus cannabinus) bast fibers during retting to identify potential candidate degumming bacteria. Retting liquids were collected and analyzed at 0 days, 10 days, and 34 days and then evaluated the yield and quality of kenaf fiber at the different retting times. Besides, the microbial communities were characterized using metagenomic and proteomic analysis by LC-MS/MS technology. RESULTS: The data showed that increase in the retting time significantly improves the softness, dispersion, and fiber whiteness of the kenaf fiber. The relative abundance of Acinetobacter increased from 2.88% at the baseline to 6.64% at the 34th retting. On the other hand, some members of Clostridium were reduced from 3% at the baseline to 2% at the 34th retting. Analysis of carbohydrate active enzymes showed constant changes in the utilization of carbohydrates. Besides, benzoquinone reductase, cellobiose dehydrogenase, glucose 1-oxidase, aryl alcohol oxidase and alcohol oxidase were the top five most abundant enzymes in the retting liquids. This present results demonstrated that the expressions of B7GYR8, Q6RYW5 and Q6FFK2 proteins were suppressed in Acinetobacter with the retting time. On the contrary, P05149 was upregulated with the retting time. In Clostridium, P37698, P52040 and P54937 proteins were upregulated with the retting time. CONCLUSION: In addition, bacteria Acinetobacter and Clostridium might be playing important roles in the kenaf degumming process. Similarly, up-regulation of P37698, P52040 and P54937 proteins is an important manifestation and mediates important roles in the degumming process.

Improvement on Thermostability of Pectate Lyase and Its Potential Application to Ramie Degumming.

In order to obtain a thermostable pectate lyase for ramie degumming, a rational design based on structural analysis was carried out on a novel pectate lyase (Pel419) derived from the Dickeya Dadantii DCE-01 for high-efficiency ramie degumming. A total of five potential amino acid sites were chosen to replace residues. Then, the mutant enzymes were subjected to the heterologous expressions in Escherichia coli and their enzymatic characteristics were determined. The optimal reaction temperature for the five mutants kept consistent with that for the wild type. The enzyme activity and thermal stability of mutant V52A were significantly improved. Meanwhile, the weight loss rate obtained by V52A with the best enzymatic characteristics in the ramie degumming process at 50 °C is comparable with that obtained by commercial cotton-ramie processing pectinases, indicating that V52A was a potential industrial enzyme that could be applied to large-scale ramie degumming. In this study, the biological functions of conservative residues of Pel419 were preliminarily explored. The mutant V52A with both enzymatic activity and improved heat resistance was acquired, providing a superior material for developing enzyme preparations of ramie degumming, and rendering an effective method for the rational design aiming to improve the thermostability of pectate lyase.

Electrochemical determination of synephrine by using nafion/UiO-66/graphene-modified screen-printed carbon electrode.

The screen-printed carbon electrode (SPCE) was one step modified with graphene (GN) and UiO-66 composites in nafion solution as a portable sensor (nafion/UiO-66/GN/SPCE) for the detection of synephrine. The used GN and UiO-66 were well-characterized, exhibiting typical structures by scanning electron microscope (SEM) with energy dispersive spectroscopy (EDS), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The nafion/UiO-66/GN/SPCE showed the maximum electrochemical signals for synephrine when comparing fabricated components in the cyclic voltammetric method. It was systematically investigated in modifier composition, modification volumes, pH, scan rate, and quantitative analysis ability. Under optimal conditions, the sensor exhibited a good detection limit (0.04 µmol L-1) for synephrine with a linear range of 0.5 µmol L-1 to 60 µmol L-1 (r2 = 0.995). The nafion/UiO-66/GN/SPCE had adequate reproducibility and stability with relative standard deviations lower than 2.01%. It was also applied to determine synephrine in the extract of Citrus aurantium L. with recoveries between 99.0% and 102.0%. The content of synephrine was in good agreement with that of the HPLC method. Based on its convenience and stability, the proposed nafion/UiO-66/GN/SPCE could be further developed as a portable and rapid detection sensor for natural active compounds in food, agricultural, and pharmaceutical fields.

Electrochemical Detection of Olivetol Based on Poly(L-Serine) Film Layered Copper Oxide Modified Carbon Paste Electrode (p-L-Serine/CuO/CPE).

Olivetol is an important polyphenol compound and intermediate in the synthesis of cannabinoids possessing many types of biological activities. A facile electrochemical sensor for olivetol was fabricated based on p-L-serine, and copper oxide (CuO) nanoparticles modified carbon paste electrode (p-L-serine/CuO/CPE). The proposed p-L-serine/CuO/CPE was applied to the electrochemical detection of olivetol by cyclic voltammetry (CV) and differential pulse voltammetric (DPV). Through the characterizations of materials and modified electrodes, the p-L-serine/CuO/CPE exhibited enhanced electrochemical signals for olivetol compared to bare CPE and CuO/CPE in both CV and DPV methods. Under the optimized conditions, the proposed p-L-serine/CuO/CPE showed a good quantitative analysis ability and a wide analysis range from 20 to 100 µmol L-1 of olivetol with a limit of detection of 1.04 µmol L-1. Based on the reproducibility, repeatability, and stability exhibited by this fabricated sensor and the cheap and accessible raw materials, the p-L-serine/CuO/CPE became a novel determination choice for olivetol in the electrochemical method with the advantages of being cost-effective and convenient.

Bacterial strain for bast fiber crops degumming and its bio-degumming technique.

The research and development of bio-degumming technology is under a slow progress due to the shortage of proper efficient bacterial strains and processes. A degumming bacterial strain-Pectobacterium wasabiae (PW)-with broad-spectrum degumming abilities was screened out in this study. After the fermentation for 12 h, the residual gum contents of kenaf bast, ramie bast, hemp bast, flax bast, and Apocynum venetum bast were all lower than 15%. This bacterial strain could realize the simultaneous extracellular secretion of pectinase, mannase, and xylanase with the maximum enzyme activity levels of 130.25, 157.58, and 115.24 U/mL, respectively. The optimal degumming conditions of this bacterial strain were as follows: degumming time of 12 h, bath ratio of 1:10, temperature of 33 °C, and inoculum size of 2%. After the bio-degumming through this bacterial strain, the COD in wastewater was below 4000 mg/L, which was over 60% lower than that in boiling-off wastewater generated by chemical degumming. This technology achieves higher efficiency, higher quality, and lower pollution.

Purification and Characterization of a Thermostable ß-Mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases.

ß-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the ß-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a "two-step" method comprising ultrafiltration and gel chromatography. The purified ß-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The ß-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 µmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, ß-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the ß-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.

Bio-degumming technology of jute bast by Pectobacterium sp. DCE-01.

Among industrial fiber crops, jute is ranked second to cotton in terms of yield and planting area worldwide. The traditional water retting and chemical semi-degumming methods restrict the development of the jute industry. Jute fiber can be extracted from jute bast through mechanical rolling (preprocessing), culture of bacteria, soaking fermentation (liquor ratio = 10, inoculum size = 1 %, temperature = 35 °C, and time = 15 h), inactivation, washing, and drying. Pectobacterium sp. DCE-01 secretes key degumming enzymes: pectinase, mannase, and xylanase, which match well the main non-cellulosic components of jute bast. Compared with the traditional water retting degumming, the bio-degumming cycle is shortened from more than 10 days to 15 h. The proposed bio-degumming achieved higher efficiency and lower pollution than water retting and chemical semi-degumming.

Construction and co-expression of polycistronic plasmids encoding bio-degumming-related enzymes to improve the degumming process of ramie fibres.

OBJECTIVES: To research the inherent properties of the co-expression of three types degumming-related enzymes and breed more powerful degumming strains. RESULTS: Six tandem multimers of the pectate lyase gene, the xylanase gene, and the endo-1,4-ß-mannanase gene, which are essential for degumming process, were co-expressed and evaluated in Escherichia coli BL21(DE3). The xyl91 gene had a synergistic effect with endo-1,4-ß-mannanase and pectate lyase from DCE-01, when xyl gene was replaced with xyl91 in the multimer. The recombinant pET-pxm(91x) was selected and transformed into the original degumming strain DCE-01, which led to an enzymatic activity improvement. Furthermore, the weight loss, reducing sugar and COD value of the sample treated with the new engineered strain pET-pxm(91x)/DCE-01 increased to 22.5 %, 460 mg ml-1 and 4.9, respectively. CONCLUSIONS: The co-expression of degumming-related enzyme genes may be applied in industrial tests and represents a novel direction for bio-degumming research.

Purification and characterization of a xylanase from Bacillus subtilis isolated from the degumming line.

Xylanases catalyze the hydrolysis of xylan, a major hemicellulose component of cell wall besides cellulose in most plant species. To extract cellulose fibers, it will be invaluable to screen for more effective xylanase-producing microorganisms. In this paper a new strategy for easy screening of xylanase-producing strains from the degumming line was presented. Using this strategy, a weak-acidic, cellulase-free xylanase from Bacillus subtilis has been isolated, purified and characterized. The xylanase showed high specific activity (36,633.4 U/mg), presented stable characteristics and can be separated and purified simply, with molecular weight 23.3 kD, pI 9.63. It reached its optimal activity at pH 5.8 and 60 °C, and retained over 80% of its maximal activity after pre-incubation at temperature 60 °C or pH 4.6 ~ 6.4. Also, a two-step purification procedure based on the combination of ultrafiltration and gel filtration chromatography was introduced and described, achieving 17-fold purification with 68.11% yield.

Sphingobacterium bambusae sp. nov., isolated from soil of bamboo plantation.

A Gram-negative, non-motile, non-spore-forming bacterial strain designated IBFC2009(T) was isolated from soil of a bamboo plantation. The strain could grow at 11 degrees C approximately 39 degrees C, pH 6.0-9.0, and in the presence of 0 approximately 5% NaCl. Based on 16S rRNA gene sequence analysis, Strain IBFC2009(T) belonged to the genus Sphingobacterium and showed the highest sequence similarity of 94.6% (S. composti T5-12(T)) with the type strains within the genus. The major fatty acids were summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1) omega7c, 34.4%), iso-C(15:0) (22.4%), C(16:0) 3-OH (15.2%), and iso-C(17:0) 3-OH (12.8%). The G+C content of the genomic DNA was 41.0 mol%. According to the phenotypic and genotypic characteristics, Strain IBFC2009(T) should represent a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bambusae sp. nov. is proposed. The type strain is IBFC2009(T) (=CCTCC AB 209162(T) =KCTC 22814(T)).