High-Level Extracellular Expression of a New ß-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc.

N-acetyl-ß-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. ß-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

[Expression and characterization of ß-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-ß-D glucosamine production].

ß-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-ß-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Biofiltration of a mixture of ethylene, ammonia, n-butanol, and acetone gases.

This study describes cleaning of a waste gas stream using bench scale biofilters (BFs) or biotrickling filters (BTFs). The gas stream contained a mixture of acetone, n-butanol, methane, ethylene, and ammonia, and was diverted uniformly to six biofilters and four biotrickling filters. The biofilters were packed with either perlite (BF-P), polyurethane foam (BF-F), or a mixture of compost, wood chips, and straw (BF-C), whereas the biotrickling filters contained either perlite (BTF-P) or polyurethane foam (BTF-F). Experimental results showed that both BFs and BTFs packed with various media were able to achieve complete removal of highly soluble compounds such as acetone, n-butanol, and ammonia of which the dimensionless Henry's constants (H) are less than 0.01. Methane was not removed due to its extreme insolubility (H>30). However, the ethylene (H ≈ 9) removal efficiencies depended on trickle water flow rates, media surface areas, and ammonia gas levels.

The effect of nitrate on ethylene biofiltration.

This study investigated the effects of filter media types and nitrate (NO(3)(-)) concentrations in nutrient solutions on C(2)H(4) biofiltration. A new nutrient solution with zero NO(3)(-) concentration was supplied to two perlite-bed biotrickling filters, two perlite-bed biofilters, and two GAC (Granular Activated Carbon)-bed biofilters, while the other with 2 g L(-1) of NO(3)(-) was used for the other two GAC biofilters. All reactors underwent a total test duration of over 175 days with an EBRT (Empty Bed Residence Time) of 30 s, inlet gas flow rate of 7 L min(-1), and inlet C(2)H(4) concentrations of 20-30 mg m(-3). NO(3)(-) concentration and media type significantly affected the C(2)H(4) removal efficiencies in all types of biofiltration. The perlite media with no NO(3)(-) achieved C(2)H(4) removal efficiencies 10-50% higher than the others. A NO(3)(-) concentration as high as 2 g L(-1) in the original nutrient solution may act as an inhibitor that suppresses the growth or activity of C(2)H(4) degraders. In addition, the perlite media resulted in higher C(2)H(4) removal efficiencies than GAC media, because the hydrophilic surface of the perlite leads to a higher moisture content and thus to favorable microbial growth.

Activated carbon load equalization of discontinuously generated acetone and toluene mixtures treated by biofiltration.

Studies were conducted to evaluate use of granular activated carbon (GAC) as a passively operated load-equalization mechanism for biofilters treating gas streams with dynamically varying (intermittent) pollutant loading. In the initial stage of research, abiotic fixed-bed sorption experiments and numerical modeling were conducted to assess the degree of load-equalization achieved by GAC columns for air flows containing intermittent loading of acetone and toluene present as single-component contaminants and as a mixture. In the subsequent stage of research, an integrated system consisting of a GAC column in series before a biofilter was used to treat a gas stream containing a mixture of acetone and toluene at influent concentrations of 430 ppm(v) and 100 ppm(v) respectively. To simulate loading conditions expected from an industrial process with intermittent operation, contaminated air was supplied 8 h/day and uncontaminated air was supplied 16 h/day. The system was operated with different empty bed contact times, as low as 2.5 s for the GAC column and 14.5 s for the biofilter. Performance of an additional, conventionally operated biofilter (i.e., without GAC load equalization system) was used as a basis of comparison. Data are presented which clearly demonstrate that passively operated GAC load-dampening systems installed in series before biofilters can lead to more uniform loading as a function of time and thereby improve biofilter treatment performance. Results also demonstrate that, because of competitive sorption, the degree of load equalization achieved for different constituents in multi-contaminant gas streams can vary markedly. A pore and surface diffusion model (PSDM) was able to accurately predict the degree of load-dampening achieved by GAC columns for single and multicomponent waste gases.