Physicochemical characterization and antioxidant activity of polysaccharides from Chlorella sp. by microwave-assisted enzymatic extraction.

Microwave-assisted enzymatic extraction (MAEE) was used for the separation of polysaccharides from micro-Chlorella. The extraction condition of MAEE was optimized by Box-Behnken design and response surface methodology. Results showed that the optimal condition for the extraction of Chlorella sp. crude polysaccharides (CSCP) was at 50°C for 2.3 h with 380 W of microwave power and 0.31% of enzyme dosage. Under the optimal extraction condition, the extraction yield of CSCP reached 0.72%. Similarly, the α-amylase modification conditions of the CSCP were also optimized, in which the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate was used as the response value. The scavenging rate of DPPH free radicals was 17.58% when enzyme dosage was 271 U/g at 51°C for 14 min. Moreover, the enzyme-modified CSCP presented a typical heteropolysaccharide mainly including glucose (48.84%), ribose (13.57%) and mannose (11.30%). MAEE used in this work achieved a high extraction yield of CSCP, which provides an efficient method for the extraction of CSCP from Chlorella sp.

Efficient synthesis of Ala-Tyr by L-amino acid ligase coupled with ATP regeneration system.

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1 mM Ala-Tyr was produced within 3 h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89 mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4 mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase.

Programming Integrative Multienzyme Systems and Ionic Strength For Recyclable Synthesis of Glutathione.

In the enzymatic cascade catalysis, it is a big challenge to construct a stable and reusable catalyst with targeted enzymes. The artificial multienzyme reactor has attracted great attention due to its potential for facilitating the performance of enzyme catalysis. In this study, we set up a reliable system that could assemble polyphosphate kinase (PPK) with bifunctional glutathione synthetase (GshF) via SpyCatcher/SpyTag to form multienzyme systems (MESs). Furthermore, MESs could assemble into nanoaggregates by altering the ionic strength, and the larger nanoaggregates could be applied in robust and reusable synthesis of glutathione (GSH). To enhance MES levels in vivo, gene duplication and different coexpression modes were performed. Finally, the optimized production of GSH and oxidized glutathione (GSSG) reached 102.6 and 6.7 mM within 2 h. Compared with the first round, the total yield only decreased by 9.4% after five continuous rounds of biocatalysis.

Characterization of Two Polyphosphate Kinase 2 Enzymes Used for ATP Synthesis.

Enzymes used for adenosine triphosphate (ATP) synthesis play important roles in energy-dependent cascade reactions in vitro. In this study, two novel polyphosphate kinase 2 (PPK2) enzymes, HbPPK2 from Hydrogenophilaceae bacterium and NdPPK2 from Nocardioides dokdonensis, were characterized for ATP synthesis with the substrate polyphosphate (polyP). The optimum temperature and pH of both purified HbPPK2 and NdPPK2 were 30 °C and 6.5. HbPPK2 and NdPPK2 retained 30% and 14% of the initial activity at 30 °C for 12 h, respectively, whereas the presence of polyP significantly enhanced the stability of enzymes. The two PPK2s preferentially catalyzed the long-chain polyP hexametaphosphate as the phosphate donor. Adenosine monophosphate could not be used by HbPPK2 and NdPPK2 to synthesize ATP, indicating that they belonged to the class I subfamily of PPK2. HbPPK2 was used for ATP regeneration to produce glutathione by a two-enzyme cascade in vitro. 47.1 ± 0.4 mM glutathione was synthesized with a productivity of 13.5 ± 0.1 mM/h. ATP was regenerated approximately 471 times in the system within 3.5 h. HbPPK2 showed potential application for ATP regeneration in cascade reaction.

Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters.

Glutathione (GSH) is an important bioactive tripeptide and widely used in food, medicine and other industries. Recently, the bifunctional glutathione synthetase, GshF, has been applied for efficient GSH production under inducible promoter. In this study, the constitutive expression of GshF from Streptococcus sanguinis (GshFss) was investigated under four different constitutive promoters. Based on previous study, five genes in Escherichia coli JM109 were deleted to eliminate the degradation of precursors and GSH. The effects of gene knockout on the constitutive expression of GshFss and GSH production were evaluated by whole cell catalysis. Finally, the engineered strain JM03Pdel1 produced 24 mM glutathione with addition of 30 mM precursors in 5-L bioreactor fed-batch fermentation. The yield of GSH based on cysteine in JM03Pdel1was reached 80% without any inducer, which was improved by 17.3% than that in the control strain.