Fusion and secretory expression of an exo-inulinase and a d-allulose 3-epimerase to produce d-allulose syrup from inulin.

BACKGROUND: This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION: This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.

Two plant growth promoting bacterial Bacillus strains possess different mechanisms in adsorption and resistance to cadmium.

Microorganisms are promising biosorbents for decontaminating cadmium-polluted soil or water systems, but the underlying remediation mechanisms are still unclear. In this study, the cadmium biosorption mechanisms and capabilities of plant growth-promoting microorganisms (Bacillus megaterium NCT-2 and Bacillus paranthracis NT1) were investigated. Batch biosorption experiments showed that the optimal biosorption conditions for B. megaterium NCT-2 and B. paranthracis NT1 were pH 6.0, a biomass dosage of 1.0 g L-1, and an initial Cd2+ concentration of 10 mg L-1, and pH 8.0, a biomass dosage of 1.0 g L-1, and an initial Cd2+ concentration of 10 mg L-1, respectively. The biosorption processes of both biosorbents were well described by the pseudo-second order kinetic model, which indicated that the biosorption of Cd2+ was mainly chemisorption. The intracellular accumulation portion of adsorbed Cd2+ in B. megaterium NCT-2 was much higher than in B. paranthracis NT1 (43.11% and 3.25%, respectively), which resulted in the lower cadmium tolerance (14 mg L-1 and 280 mg L-1, respectively) and higher cadmium removal efficiency (46.79% and 20.45%, respectively) of B. megaterium NCT-2 compared to B. paranthracis NT1. SEM-EDS and FTIR analysis suggested the probable interactions of Cd2+ with the biosorbent surface ligands, such as -OH, -NH, -SO3, CO and -COOH during surface adsorption. Results of qRT-PCR illustrated that the difference in cadmium resistant mechanism and adsorption performance between B. megaterium NCT-2 and B. paranthracis NT1 may be regulated by the genes cadA, zitB, khtT, and bshA and cadA, trkA, czcD, and bshA, respectively. Our results revealed that these two biosorbents have the potential for further use in the development of cadmium remediation technologies and could provide insight into the mechanisms of cadmium biosorption.

Efficient Biosynthesis of Low-Molecular-Weight Poly-γ-glutamic Acid Based on Stereochemistry Regulation in Bacillus amyloliquefaciens.

Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has attracted much attention because of its many potential applications in food, agriculture, medicine, and cosmetics. Enzymatic degradation is an efficient way for the synthesis of LMW-γ-PGA. However, the stereochemistry of γ-PGA limits the degradation of γ-PGA. This study identifies the role of γ-PGA synthase (pgsA) and glutamate racemase (racE) in the regulation of γ-PGA stereochemistry and demonstrates their combinational use for LMW-γ-PGA synthesis. First, the expression of pgsA and racE was enhanced, leading to improvements both in the molecular weight (Mw) and the d-glutamate proportion of γ-PGA. Then, an optimal combination of pgsA, racE, and γ-PGA hydrolase pgdS was constructed by exchanging the gene origins for the synthesis of LMW-γ-PGA. Finally, the Mw of γ-PGA was decreased to 6-8 kDa, which was much lower compared with the case without stereochemistry switching (20-30 kDa). This study provides a novel strategy to control the Mw of γ-PGA based on stereochemistry regulation and lays a solid foundation for synthesis of LMW-γ-PGA.

Highly Chemo-, Regio- and E/Z-Selective Intermolecular Heck-Type Dearomative [2 + 2 + 1] Spiroannulation of Alkyl Bromoarenes with Internal Alkynes.

Described herein is a palladium-catalyzed dearomative annulation of alkyl bromoarenes with internal alkynes. Challenges in this spiroannulation include the chemoselectivity among [2 + 2 + 1], [2 + 2 + 2], and [3 + 2] annulations and the E/ Z-selectivity associated with the generated exocyclic double bond. In the presence of Pd(OAc)2 and a phosphine ligand, a variety of highly functionalized spirocyclopentadienes with an exocyclic carbon-carbon double bond are provided in good to excellent yields with high chemo-, regio-, and E/ Z-selectivity via a Heck-type pathway.