Oligopeptide hydrogels for localized protein delivery have considerable potential to reduce systemic side effects but maximize therapeutic efficacy. Although enzyme catalysis to induce formation of oligopeptide hydrogels has the merits of unique regio- and enantioselectivity and mild reaction conditions, it may cause the impairment of function and activity of the encapsulated proteins by proteolytic degradation during gelation. Here we report a novel enzyme-catalysis strategy for self-assembly of oligopeptide hydrogels using an engineered protease nanocapsule with tunable substrate selectivity. The protease-encapsulated nanocapsule shielded the degradation activity of protease on the laden proteins due to the steric hindrance by the polymeric shell weaved around the protease, whereas the small-molecular precursors were easier to penetrate across the polymeric network and access the catalytic pocket of the protease to convert to the gelators for self-assembling hydrogel. The resulting oligopeptide hydrogels supported a favorable loading capacity without inactivation of both an antiangiogenic protein, hirudin and an apoptosis-inducing cytokine, TRAIL as model proteins. The hirudin and TRAIL coloaded oligopeptide hydrogel for combination cancer treatment showed enhanced synergistic antitumor effects both in vitro and in vivo.Keywords: combination cancer therapy; enzyme catalysis; oligopeptide hydrogel; Protein delivery; self-assembly;

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na+ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO3. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na+-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na+-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

A glycosyltransferase GTBP1 from Bacillus pumilus BF1 was isolated. Efficient production of ponasterone A was achieved by the recombinant E. coli/gtBP1 in a biphasic system with a molar yield of 92.7%. This in situ product removal provided the “driving force” for shifting the reaction equilibrium towards the synthesis of the product.

•A novel strategy was established to improve substrate tolerance of β-galactosidase.•Hydrophilic solvent was employed to increase the stability of BMG in vanillyl alcohol solution.•0.85–2.44 g of natural glycosides were successfully obtained in 100 mL-scale.Aromatic alcohols are liable to result in enzyme inactivation. A novel strategy was established for improving the aromatic alcohol tolerance of β-galactosidase (BMG) from Bacillus megaterium YZ08. The half-life of BMG in 200 mM vanillyl alcohol solution was dramatically increased by 9–123 times with the addition of hydrophilic solvents. In 30% DMSO, the reaction concentration of aromatic alcohol could reach up to 300–400 mM, and 0.85–2.44 g of natural glycosides were successfully obtained in 100 mL-scale. The simple and effective strategy shows potential applications when dealing with the preparative-scale exploitation of enzymatic reactions.Download high-res image (211KB)Download full-size image

To improve the stability of β-galactosidase from Bacillus megaterium YZ08 (BMG) in aqueous hydrophilic solvents and promote its application in the galactosylation of natural products.The addition of 5 mM Mg2+ significantly enhanced the stability of BMG in aqueous hydrophilic solvents, and the half-lives of BMG in these solutions reached 56 min to 208 h, while they were only 7 min to 5.9 h without addition of Mg2+. Studies on the kinetic parameters in buffer solution and 30% dimethyl sulfoxide (DMSO) indicated that the affinity of BMG to 2-nitrophenyl-β-d-galactopyranoside and its catalytic efficiency (κcat/Km) increased with the addition of Mg2+. Furthermore, the addition of Mg2+ facilitated galactosylation reactions in 30% DMSO and increased product conversions by 24–41% due to the reversal of the thermodynamic equilibrium of hydrolysis.A convenient approach was established to improve the stability of BMG in aqueous hydrophilic solvents.

To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining.BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2′-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61–84%.An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.Keywords: Escherichia coli, arabinose; extracellular expression; metalloprotease; Z-aspartame;

Efficient synthesis of unitary β-galactosyl nucleoside analogues was successfully achieved in a 10% DMSO solvent system, catalyzed by a newly isolated solvent-stable β-galactosidase from solvent-tolerant Bacillus megaterium YZ08. The most efficient sugar donor for the galactosylation of nucleoside analogues was lactose rather than nitrophenyl glycoside. However, it was accompanied with the formation of complicated digalactosyl nucleoside analogues in the buffer system. The addition of DMSO not only prominently increased the stabilization of β-galactosidase but also regulated the products from complicated products to unitary monogalactosyl nucleoside analogues, which could significantly simplify the process of subsequent separation. Strikingly, the affinity of β-galactosidase to 3′-azido-3′-deoxythymidine in 10% DMSO was significantly enhanced, and the catalytic efficiency (kcat/Km) was doubled as compared to that in buffer solution. These results indicated that solvent-stable glycosidase possesses attractive potency in biosynthesis as well as in regulation of the glycoside prodrugs by adjusting the reaction solvent system.

Using both polar and low polar organic solvents (DMSO and toluene) as screening stress, a solvent-stable bacterium Burkholderia cepacia RQ3 was newly isolated. An organic solvent-stable lipase from strain RQ3 was purified in a single step with 50.1 % recovery by hydrophobic chromatography. The purified lipase was homogenous on SDS-PAGE and had an apparent molecular mass of 33 kDa. The gene of lipase RQ3 with an open reading frame of 1095 bp encoding 364-amino acid residues was cloned. The optimal pH and temperature for lipase activity were 9.0 and 40 °C. The lipase was stable in a wide pH range of 6.0–10.0 and at temperature below 50 °C. Strikingly, all the tested hydrophilic and hydrophobic organic solvents significantly extended the half-life of lipase RQ3 compared with that in a solvent-free system, which indicated that lipase RQ3 showed a broad solvent tolerance to various organic solvents. The lipase demonstrated excellent enantioselective transesterification toward the (S)-1-phenylethanol with a theoretical conversion yield of 50 % and eep of 99.9 %, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50 °C and pH of 9.0. Meanwhile, the lipase was stable below or at 30 °C and over an alkaline pH range (7.5–11.0). Ca2+ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25 %, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.

Solvent-exposed acidic/amide residue (Asp/Glu or Asn/Gln) exerts great effects on the thermostability of protein; however, experimental attempts appear to be time-consuming, so a more scientific, simple, and effective rational strategy is necessary. In this study, molecular dynamic (MD) simulation was performed to analyze two surface acidic residues (Asp37 and Glu119) of thermolysin (TLN) in mediating its thermostability. Root-mean-square-deviation (RMSD) was calculated to evaluate the thermosensitivity effect by acidic/amide substitutions. The wild-type TLN and three mutants (TLM1, TLM2, and TLM) presented significantly different thermostability effect. Four profiles of RMSD values demonstrated that the thermal insensitivity of variants were TLM2 > TLM > TLN > TLM1. As expected, the thermostability and half-life (at 60 °C) behavior of enzyme variants showed the same trends with the computational predictions, and it was worth noting that the half-life of TLM2 showed 3.1-fold longer than that of wild-type. The Tm and T50 of TLM2 were 9 and 7 °C higher, respectively, than that of wild-type enzyme. Rational substitution of acidic/amide residue in regulation of thermostability using MD simulation would be an efficient approach for instructional design to improve the thermostability.

An organic solvent-tolerant β-fructofuranosidase (β-FFase) from Arthrobacter arilaitensis NJEM01 was purified, characterized, cloned, and overexpressed in Escherichia coli. The mature β-FFase contained 495 amino acid residues with an estimated molecular mass of 55 kDa. The purified β-FFase from strain NJEM01 was very stable in the buffer systems (pH 5.0–9.5) and showed high stability below 45 °C. Furthermore, the enzyme exhibited relatively high solvent stability in various aqueous organic mixtures and retained nearly 100% of its initial activity after incubation for 10 days in 20% (v/v) DMSO. In addition, the β-FFase exhibited high transfructosylation activity, synthesized prebiotic products of mainly 6-kestose (up to 476 g/L), and showed fructosyl receptor specificity to C-glucosyl flavone. A relatively high yield of FOS was achieved by the β-FFase from bacterium with a high concentration of sucrose. It made the β-FFase an exploitable biocatalyst for the production of glycosides of natural products and prebiotic kestose.

•Z-Aspartame was highly efficient synthesized in low pH aqueous medium.•High yield of Z-APM was achieved due to “in situ product removal”.•Rationalized the inhibition by Z-l-Asp at a relatively higher concentration using molecular docking approach.Z-Aspartame (Z-APM) was obtained using an aqueous reaction at pH 6.0 based on thermodynamic control and in situ product removal. The lower pH reaction medium led to higher solubilities of the two substrates, l-PheOMe and Z-l-Asp and dramatically lowered the solubility of the product. The higher pH stability of the protease PT121 and the mutant Y114S enabled the pH modulation of the reaction medium to shift the thermodynamic equilibrium toward product synthesis. The reaction-separation coupling provided a “driving force” for the enzymatic synthesis and resulted in high yields of 88.5% (CZ-l-Asp = 50 mM, Cl-PheOMe = 500 mM) and 82.2% (CZ-l-Asp = 100 mM, Cl-PheOMe = 500 mM), without further purification for removing protection group, of Z-aspartame. Moreover, we rationalized the inhibition by Z-l-Asp at a relatively higher concentration on the synthesis of Z-APM using a molecular docking approach.Download full-size image