High-level production of G protein-coupled receptors (GPCRs) is usually difficult to achieve in heterologous cell systems. The inherent hydrophobicity of these receptors could cause aggregation and possible cytotoxicity. Cell-free (CF) expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. Here we reported the CF production of an olfactory receptor from Caenorhabditis elegans, odorant response abnormal protein 10 (ODR-10), a member of GPCRs, using the Escherichia coli extracts. Different expression vectors were investigated and 175 μg/ml total ODR-10 was achieved with pIVEX2.4c. To obtain soluble ODR-10, different detergents and liposome with varied concentrations were respectively added into the CF system. High-level expression of soluble ODR-10 (150 μg/ml) was attained with the addition of 1.5 % polyoxyethylene-(20)-cetyl-ether (Brij58) into the CF system. Furthermore, the yield of total ODR-10 was improved to 350 μg/ml by supplementing liposomes into the CF system, and the maximal concentration of the soluble receptor (102 μg/ml) was achieved in this liposome-assisted CF system. Both strategies produced ODR-10 efficiently by using CF system, and the direct reconstitution of the in vitro expressed receptor into liposomes will be preferred for its potential applications in many areas.

A batch method was applied to investigate the adsorption behavior and mechanisms of L-tryptophan (L-trp) on ion exchange resins. HZ-001 and JK006 were proved to be ideal adsorbents due to their large loading capacityand favorable selective adsorption for L-trp. Langmuir, Freundlich, and Dubinin-Radushkevich equations were appliedto simulate the experimental data to describe the adsorption process of L-trp onto HZ-001 and JK006. The maximumloading capacity (at pH 5.0, 30 °C), determined by the Langmuir and Dubinin-Radushkevich models, was close to eachother (833 mg/g vs. 874 mg/g) for HZ-001, while discrepant (833 mg/g vs. 935 mg/g) for JK006. Three diffusion-controlled kinetic models were utilized to analyze the results in order to identify the adsorption mechanism. The adsorptionkinetics of L-trp onto cation exchange resins was investigated under different experimental conditions, including initialsolution pH, temperature, initial L-trp concentration, and adsorbent dosage. Moreover, the diffusion process of L-trponto HZ-001 and JK006 was evaluated at different initial adsorbate concentrations. The thermodynamic parameters, obtained from the kinetic data, demonstrated that L-trp could be adsorbed spontaneously onto both resins.

The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L−1) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.

A novel method involving polyethylenimine (PEI) coating and glutaraldehyde cross-linking processes was developed to immobilize Candida rugosa lipase onto cotton cloth. After the systematic investigation, the optimal lipase immobilization was achieved when 0.1 g lipase was loaded on 1.5 g support, which was pretreated with 10 ml of 1.0 mg/ml PEI solution at pH 8.0. Subsequent catalytic analysis of immobilized lipase for ethyl butyrate synthesis was also carried out in the Erlenmeyer flasks. The results indicated that when optimal 0.25 M ethanol and 0.6 M butyric acid were catalyzed by the immobilized lipase at 25 °C, the highest conversion yield of 91.2% and 1.27 mmol h−1 g−1 productivity of ethyl butyrate were obtained. Furthermore, a kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was proposed and validated by experimental data. To explore the practical potential of immobilized lipase, its operational stability was evaluated and the residual activity was remained about 50% after 12 repeated recycles, with a half-life time of about 300 h for the immobilized lipase. Finally, a recycle batch reactor using immobilized lipase was developed for ethyl butyrate production. The achieved result of 0.85 M final ethyl butyrate concentration, with the conversion of 70.6% and the productivity of 1.45 mmol h−1 g−1, had revealed the promising potential of this immobilized lipase in practical applications.Graphical abstractDownload full-size imageHighlights► Candida rugosa lipase was immobilized onto cotton cloth. ► The effects of PEI, pH value and the amount of support were investigated to improve the immobilization efficiency. ► The half-life time of the immobilized lipase is about 300 h. ► Ping Pong Bi-Bi mode with inhibition of substrates was proposed and validated. ► A recycle batch reactor using immobilized lipase was established.

•PQQGDH was evolved by multiplex sites in situ engineering for enhanced thermal stability and substrate specificity.•A color reaction-based high-throughput screen method was constructed to analyze mutants.•Two variant proteins showed enhanced substrate specificity and thermal stability than the parent PQQGDH enzyme.Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) is a novel oxygen-independent oxidoreductase. In this work, the PQQGDH gene was integrated into the chromosome of Escherichia coli EcNR2 and then modulated by multiplex-site in situ engineering to achieve improved substrate specificity and thermal stability. After four mutagenesis cycles executed within a single day, six PQQGDH variants were selected and characterized. The variants Ser231Lys/Asn452Thr or Ser231Lys showed higher stability relative to the wild-type enzyme. Additionally, the wild-type PQQGDH could only utilize maltose and lactose at 60% efficiency relative to glucose, while four variants (Ser231Cys/Asn452Thr, Ser231Lys/Asn452Thr, Asn452His, and Asn452Thr) showed significant reductions in their ability to utilize both maltose (30%) and lactose (45%). In particular, Ser231Cys/Asn452Thr and Ser231Lys/Asn452Thr possessed both improved substrate specificity and thermal stability compared to the wild-type PQQGDH. Our study provided a novel and efficient approach to simultaneously introduce multiplex mutations in situ, which is a potential strategy for target enzyme evolution in vivo.Download full-size image

With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β-d-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of rabavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.

•Several leader peptides were fused to increase AqpZ expression in cell-free system.•Leader peptide sequences could be in situ removed by adding detergents or liposome.•The purified AqpZ from these cell-free systems was fully bioactive.Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10−14 cm3 s−1 monomer−1. Using this in situ removable leader peptide strategy, the transcription–translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.

Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with many potential applications. Although microbial fermentation is the common approach to produce γ-PGA, the broth at the latter stage usually becomes very viscous and severely influences the metabolites producing pattern of target microbe. In this study, acetoin and 2,3-butanediol were confirmed to be the main byproducts of γ-PGA-producing strain B. subtilis ZJU-7 (B. subtilis CGMCC1250), and their effects on the cell growth and γ-PGA biosynthesis were further investigated in shake flasks. The outcome indicated that both acetoin and 2,3-butanediol showed clear impairment on γ-PGA production of B. subtilis ZJU-7. Moreover, the extracellular metabolites profiles of fermentation under three different pH values were acquired and the metabolic flux redistribution of pathways related to γ-PGA biosynthesis was calculated based on the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at pH 6.5, in which the ingestion rate of extracellular glutamic acid was higher and the subsequent γ-PGA biosynthesis was enhanced. The present work provided us a deep insight into the metabolic flux control of γ-PGA biosynthesis, which will stimulate some novel metabolic engineering strategies to improve the productivity of γ-PGA in future.Highlights► Acetoin and 2,3-butanediol were confirmed as the main byproducts in B. subtilis ZJU-7. ► Extracellular metabolite profiles of PGA fermentation were acquired at three pH values. ► Pathways of byproducts were included in metabolic network modeling of PGA biosynthesis. ► Metabolic flux redistribution of PGA and byproducts formation was calculated.