Enhanced enzymatic sugar production from corn stover by combination of water extraction and glycerol-assisted instant catapult steam explosion.

Glycerol-assisted instant catapult steam explosion (ICSE) of lignocellulose is an effective pretreatment method for enhancing sugar production compared to glycerol-free ICSE. In this study, glycerol-assisted ICSE of corn stover was studied in order to understand the reaction mechanisms and further optimize the process. Results showed that water extraction of corn stover prior to ICSE reduced pseudo-lignin formation. The combination of water extraction and glycerol-assisted ICSE led to the formation of lignin with a lower molecular weight (Mw) of 2851 g/mol than 3521 g/mole of that from the combination of water extraction and glycerol-free ICSE. 1H-13C NMR analysis revealed that glycerol likely reacted with lignin carboxylic OHs through esterification while etherification of aliphatic OHs was not observed in ICSE. These lignin analyses indicated that glycerol protected lignin from condensation/repolymerization during glycerol-assisted ICSE. Enzymatic hydrolysis results showed that without water extraction increasing glycerol usage from 0.2 kg/kg stover to 0.4 kg/kg stover improved glucan digestibility to 78% but further increase to 0.5 kg/kg stover reduced glucan digestibility. In addition, at the glycerol usage of 0.2-0.4 kg/kg stover, washing of pretreated stover for removal of glycerol and other biomass-derived compounds did not improve glucan digestibility compared to unwashed ones. Combination of water extraction and glycerol-assisted ICSE led to a high glucan digestibility of 89.7% and a total glucose yield of 25.5 g glucose/100 g stover, which were 30.1% and 7.5 g/100 g stover higher than those derived from glycerol-free ICSE of stover, respectively. Since glycerol is a low-cost carbon source, the resulting enzymatic hydrolysate that contained both glucose and glycerol may be directly used to produce bioproducts by microbial fermentation.

Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch.

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182-7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin's phosphorolysis.

Detoxication and bioconversion of aflatoxin B1 by yellow mealworms (Tenebrio molitor): A sustainable approach for valuable larval protein production from contaminated grain.

Yellow mealworm (Tenebrio molitor) is a supplementary protein source for food and feed and represents a promising solution to manage grain contaminated with Aflatoxin B1 (AFB1). In this study, AFB1 present in different concentrations in wheat bran was treated and removed via bioconversion by yellow mealworm of different instars, with emphasis on the bioconversion performance and metabolism of AFB1. Upon application of wheat bran spiked with 100 µg/kg AFB1 to 5th-6th instar yellow mealworms, the conversion rate of AFB1 was up to 87.85 %. Low level of AFB1 (< 2 µg/kg) was accumulated in the larval bodies, and the survival rate, development and nutrition contents of yellow mealworm were not significantly affected. It was revealed that 1 kg of wheat bran contaminated with AFB1 increased the weight of yellow mealworms from 138 g to 469 g, containing approximately 103 g of protein. The bioconversion of AFB1 by yellow mealworms led to generation of 13 metabolites in the frass and 3 metabolites in the larvae. AFB1 was detoxicated and removed via phase I metabolism comprising reduction, dehydrogenation, hydration, demethylation, hydroxylation, decarbonylation and ketoreduction, followed by phase II metabolism involving conjugation of amino acid, glucoside and glutathione (GSH). The toxicity of AFB1 metabolites was deemed lower than that of AFB1 according to their structures. This study provides a sustainable approach and theoretical foundation on using yellow mealworms for cleaner grain contamination management and valuable larval protein production via bioconversion of food and feed contaminated by AFB1.

Improvement of Nicotine Removal and Ethanol Fermentability From Tobacco Stalk by Integration of Dilute Sulfuric Acid Presoak and Instant Catapult Steam Explosion Pretreatment.

The nicotine from tobacco stalk showed obvious inhibitory effect on the activity of cellulase and fermentability of microorganisms, which seriously hinders the utilization of tobacco stalk. Dilute sulfuric acid presoak of tobacco stalk was used to enhance the performance of instant catapult steam explosion (ICSE) for tobacco stalk pretreatment. The presoak was beneficial to break the recalcitrant structure of tobacco stalk, reduce nicotine content to relieve the inhibition on the activity of cellulase and metabolism of microorganisms, and promote the performance of enzymatic hydrolysis and ethanol fermentation. The optimized 0.8% sulfuric acid (w/w) presoak-integrated ICSE pretreatment resulted in 85.54% nicotine removal from tobacco stalk; meanwhile, the total sugar concentration from enzymatic hydrolysis of pretreated tobacco stalk increased from 33.40 to 53.81 g/L (the ratio of dry tobacco stalk to water was 1:8, w/w), ethanol concentration increased 103.36% from 5.95 to 12.10 g/L in flask, compared with separate ICSE pretreatment. Finally, the ethanol concentration achieved the highest 23.53 g/L in a 5-L fermenter with the ethanol yield from the glucose of tobacco stalk hydrolysate achieving 71.40% by increasing the solid loading of the tobacco stalk in the enzymatic hydrolysis process (the ratio of dry tobacco stalk to water was 1:4, w/w). These results achieved the expected purpose of efficient utilization of discarded tobacco stalk.

An Engineered Thermostable Laccase with Great Ability to Decolorize and Detoxify Malachite Green.

Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac) was cloned and expressed in Escherichia coli. Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays. In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.

Reassessment of the role of CaCO3 in n-butanol production from pretreated lignocellulosic biomass by Clostridium acetobutylicum.

In this study, the role of CaCO3 in n-butanol production was further investigated using corn straw hydrolysate (CSH) media by Clostridium acetobutylicum CICC 8016. CaCO3 addition stimulated sugars utilization and butanol production. Further study showed that calcium salts addition to CSH media led to the increase in Ca2+ concentration both intracellularly and extracellularly. Interestingly, without calcium salts addition, intracellular Ca2+ concentration in the synthetic P2 medium was much higher than that in the CSH medium despite the lower extracellular Ca2+ concentrations in the P2 medium. These results indicated that without additional calcium salts, Ca2+ uptake by C. acetobutylicum CICC 8016 in the CSH medium may be inhibited by non-sugar biomass degradation compounds, such as furans, phenolics and organic acids. Comparative proteomics analysis results showed that most enzymes involved in glycolysis, redox balance and amino acids metabolism were up-regulated with CaCO3 addition. This study provides further insights into the role of CaCO3 in n-butanol production using real biomass hydrolysate.

Availability of lignocellulose from forestry waste for use as a biofuel in China.

Biomass is a very important renewable energy and plays an important role in the energy structure of China. Here, the role of forestry waste in producing energy in China was analyzed and the availability of forestry waste for biofuel production, theoretically collectable amounts of forest biomass, and density of forestry waste were assessed. Agricultural and forestry waste are important biomass resources. The potential for using forestry waste as a low cost substrate for producing fuel ethanol using existing forestry resources and techniques was analyzed, and the feasibility of producing fuel ethanol in different Chinese provinces was assessed using the specific situation for each province. The results showed that 1081.73 × 106 t of forestry waste could be produced in China, and 270.43 × 106 t (25% of the amount that could be collected) could be used to produce fuel ethanol. Assuming 10 t of sawdust could be converted into 1 t of ethanol, 27 × 106 t of ethanol could be produced from forestry waste. Different provinces have different potentials for producing ethanol from forestry waste, Guangdong Province, Guangxi Province, Sichuan Province, and Yunnan Province having higher potentials than the other provinces. It was predicted that 4478 × 106 t of fuel ethanol could be produced from woodcraft waste by 2020, and the provinces with the most potential were found to be Fujian Province, Heilongjiang Province, Jilin Province, Shanxi Province, Sichuan Province, Xinjiang Province, and Yunnan Province. Using forestry waste to produce ethanol could alleviate the energy shortage in China.

Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

Crystal structure of E. coli lipoprotein diacylglyceryl transferase.

Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer.

Crystal structure of the E. coli peptide transporter YbgH.

E. coli YbgH belongs to the family of proton-dependent oligopeptide transporters (POTs), a subfamily of the major facilitator superfamily (MFS) of secondary active transporters. Like other MFS transporters, POT proteins switch between two major conformations during substrate transport. Apart from possessing a canonical 12-helix, two-domain transmembrane (TM) core, prokaryotic POT proteins usually have two TM helices inserted between the two domains. Here we determined the crystal structure of YbgH in its inward-facing conformation. Our structure-based functional studies investigated the roles of both the POT signature motif 2 and the inserted interdomain TM helix pair in the stabilization and regulation of the major conformational change in MFS/POT transporters. Furthermore, of all the proton-titratable amino acid residues, Glu21 is the only conserved one (among POTs) located in the central cavity and is critical for in vivo transport. Together, our results support the notion that MFS symporters utilize a transport mechanism based on substrate-protonation coupling.