Biochemical Engineering Journal最新文献

Cortisol, the primary glucocorticoid in humans, plays crucial physiological functions and serves as an intermediate for synthesizing other glucocorticoids. Currently, cortisol production mainly relies on a semi-synthetic route, where the key step of introducing 11β-OH into 11-deoxycortisol is catalyzed by the filamentous fungi Curvularia lunata and Absidia orchidis. This method, however, generates by-products and involves lengthy cultivation. To achieve specific and efficient production of cortisol, we constructed a recombinant biocatalyst by expressing and engineering the human mitochondrial 11β-hydroxylase CYP11B1 in Escherichia coli. Firstly, the balance between CYP11B1 and its redox partners AdR and Adx was regulated through ribosome binding site (RBS) engineering, resulting in a slight increase in cortisol productivity (from 344±19 mg·L⁻¹·d⁻¹ to 407±7 mg·L⁻¹·d⁻¹). Subsequently, the heterologous expression of CYP11B1 was improved through application of the computational design tool PROSS, generating a triple mutant S169V/H354D/L463F with 87.5 % higher cortisol yield than the wild type. Finally, the catalytic performance was improved by optimizing the recombinant protein expression conditions and enhancing the substrate solubility in the reaction system, further elevating the productivity of cortisol to 2.8±0.1 g·L⁻¹·d⁻¹. To our knowledge, this is the highest ever reported cortisol productivity using a human 11β-hydroxylase-based biocatalyst.

Plasma fractionation stands as a pivotal process for the production of therapeutic and diagnostic proteins, such as albumin and immunoglobulin G. Besides these two primary proteins in human plasma, numerous other proteins can be purified for therapeutic purposes. To support process development, a flowsheet modeling-based approach is utilized to improve production efficiency and productivity while minimizing the resource investments. The flowsheet model is first built to represent the baseline drug substance production process at pilot-scale, with operating parameters extrapolated from lab-scale experiments conducted at CSL Behring. To improve operational efficiency and save costs, throughput analysis is applied to enhance the batch throughput through new process design, scheduling, and bottleneck identification. Through implementing the strategies, the batch throughput could be increased by 47.2 % by introducing one additional operator and one buffer preparation tank into the process. Furthermore, after applying a new strategy involving multiple extractions of the initial material (paste), the batch throughput was doubled, with operating cost of goods reduced by 36.1 %. To assess the performance of the modified design and validate the model results, the pilot-scale experiments with two extractions were performed by CSL Behring and compared with model predictions, resulting in good agreement. This work demonstrates the potential of flowsheet modeling in facilitating process development from lab-scale to pilot-scale, fostering cost-effective and efficient production with limited resource investment.

The polyhydroxyalkanoate terpolymer, P[(3HB)-co-(3HV)-co-(4HB)], is a promising plastic alternative for specialized applications, notably in medical and pharmaceutical sectors. Haloferax mediterranei (Hfx), an extreme halophile archaeon, is a P[(3HB)-co-(3HV)-co-(4HB)] terpolymer production host, however the native molar proportion of 4HB incorporated into the terpolymer is low. To improve incorporation, four 4-hydroxybutyrate-CoA transferases/synthetases from Clostridum kluyveri (OrfZ), Clostridium aminobutyricum (AbfT), Nitrosopumilis maritimus (NmCAT), and Cupriavidus necator N-1 (CnCAT), were heterologously expressed in H. mediterranei, and evaluated for their ability to supply 4HB-CoA for PHA terpolymer production. Growth, PHA synthesis, and polymer composition were evaluated for the four heterologous strains in shake-flask, with Hfx_NmCAT demonstrating superior growth, terpolymer titre and 4HB molar ratio. Co-feeding with γ-butyrolactone was optimised, and Hfx_NmCAT was further evaluated under fed-batch fermentation where a maximum PHA titre of 0.7 g/L, containing 52 mol% 4HB, was achieved. This is an order of magnitude improvement in 4HB terpolymer incorporation by H. mediterranei.

Phospholipase D (PLD) is essential for the bioconversion of phosphatidylcholine (PC) to phosphatidylserine (PS), a process valuable in functional food and medicine. This study explores the stability and catalytic properties of PLD immobilized on chitosan-encapsulated magnetic nanoparticles (CMNPs), utilizing oxidized dextran (DX) and glutaraldehyde (Glu) as cross-linkers. The cross-linker concentration and immobilization time were optimized to assess their effects on PLD catalytic performance. PLD immobilized on CMNPs with DX (DX-CMNPs-PLD) exhibited optimal activity at pH 8.0 and 30 °C, retaining over 40 % activity after 14 cycles, while Glu-cross-linked PLD (Glu-CMNPs-PLD) retained approximately 65 %. DX-CMNPs-PLD demonstrated superior pH, temperature, and operational stability compared to free PLD. Additionally, the immobilized PLD was characterized using transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. Kinetics parameters (V_max and K_m) of the immobilized PLD were also studied with free PLD serving as a control. Conformational analyses indicated a significant change in PLD's secondary structure, particularly in β-sheet content, which likely contributed to the enhanced stability and activity. These findings suggest a promising approach for PLD immobilization on CMNPs, with notable implications for biotechnological applications.

Enterobacter cloacae G, a novel denitrifying phosphorus-accumulating bacterial strain, was isolated from anaerobic sludge tank of a wastewater treatment plant used for pig farms. It was discovered that a pH of 7, a temperature of 30°C, an initial phosphorus concentration of 8 mg/L, and a C/N ratio of 10 were the strain's ideal growth conditions. To ensure the stability of strain G in wastewater treatment, strain G was immobilized by 5 % polyvinyl alcohol, 2 % sodium alginate, and 0.6 g of biochar and crosslinked for 9 h in 4 % calcium chloride saturated boric acid solution via an orthogonal test. After the immobilized microspheres were introduced into the sequencing batch reactor (SBR), the nitrate and phosphate removal rates achieved were 89.36 % and 65.53 %, respectively, with a hydraulic retention time (HRT) of 8 hours, a pH of 7.5, and a C/N ratio of 4.5. The immobilized microspheres containing strain G demonstrated potential for the treatment of nitrogen-rich and phosphorus-rich wastewater.

Biomethanation offers a promising route for the valorization of synthesis gas, yet significant challenges arise from the limited conversion of carbon monoxide (CO). This study investigated the adaptation of an anaerobic microbiome in a continuous trickle bed reactor (TBR) with CO as the sole carbon and energy source. We evaluated reactor performance and microbial community changes under different CO loading rates and gas retention times (GRT). Optimal performance was achieved at a CO loading rate of 5.16 Nm³ m⁻³ d⁻¹ and a GRT of 60.6 min, resulting in average production rates of 0.99 Nm³ m⁻³ d⁻¹ for CH₄ and 2.55 Nm⁻³ m⁻³ d⁻¹ for CO₂, with an 88 % CO conversion rate. Microbial analysis indicated that the community was dominated by the genus Methanothermobacter, known for its ability to utilize CO as a sole substrate, followed by a co-dominance of syntrophic acetate-oxidizing bacteria Syntrophaceticus. This syntrophic relationship between Methanothermobacter and Syntrophaceticus is expected to be crucial for the efficient CO conversion process. Additionally, the study proposes a two-reactor system for converting synthesis gas to grid-quality methane.

Laccases are versatile biocatalysts with interest for various industrial applications. This study reports the expression of Trametes versicolor laccase in Komagataella phaffii X33. The cultivation parameters (methanol and CuSO₄ concentration, and temperature) for recombinant laccase production were studied in an orbital shaker. Enhanced laccase production was achieved by adding 1 % (v/v) methanol daily, supplementing 0.1 mM CuSO₄ and incubating at 25 °C. Under these conditions, laccase production was scaled-up in a 4 L stirred tank bioreactor. Subsequently, laccase was concentrated and purified using a combined protocol of ultrafiltration and acetone precipitation, achieving a purification factor of 3.02. The laccase produced exhibited robust stability within a pH range from 4.0 to 8.0 and thermal stability up to 30 °C. Michaelis Menten kinetic revealed Michaelis constant (K_M) and maximum rate of reaction (V_max) values of 44.5 µM and 110.9 µM/min, respectively. Finally, laccase was employed as a biocatalyst to assist the polymerization of dopamine to polydopamine, allowing the one-step coating of cellulose filter paper, as confirmed by diffuse reflectance spectroscopy (UV-Vis DRS) and scanning electron microscopy (SEM). This work represents an advance in the field of laccase production in both orbital shaker and bioreactor, while demonstrating, for the first time, the laccase-assisted polymerization of dopamine for the coating of filter paper with polydopamine.

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non-ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, our group isolated a B. subtilis strain F6 with a fast response time for mannanase production upon exposure to PKM. This work focuses on improving the mannanase production of the B. subtilis strain to achieve greater fibre hydrolysis of PKM without extending fermentation time. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was homologously expressed using a replicative plasmid (pBE-S). Enzyme production was systematically improved by optimizing various regulatory elements, including the promoter, ribosome binding site, and signal peptide. Consequently, the neutral detergent fibre content of PKM was substantially reduced by 36.4 % in 22 h of solid-state fermentation using the engineered strain. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization.

Microalgae grabbed the attention worldwide because of their application in renewable energy with a number of environmental benefits such as carbon dioxide assimilation to produce biofuel. In this study, a kinetic model aiming to analyze algae growth and lipid production was developed. Biomass was divided into two parts, algae residual cell and lipid, to analyze their kinetics distinctively and to find out the inside process mechanism. The model was calibrated and validated with different experimental datasets, varying carbon sources, and phosphate concentrations. The capability of the model to predict the dynamics of algal culture over a broad range of growth conditions was investigated. The presence of acetate reduced the bicarbonate uptake for algal growth and growth on organic carbon was higher compared to that of carbon dioxide. The presence of ammonium showed a very strong inhibition effect on algae and lipid production rate but enhanced lipid content. Phosphate caused both limitation and inhibition effects on algae growth and lipid production rate. The maximum growth was found at 12 mg/L PO₄^3- concentration and 3.10 mg/L NO₃^--N concentration. Lipid content was enhanced by limiting phosphate. Overall, the developed model allows optimizing of nutrient concentrations and operating conditions specifically to enhance lipid productivity.

Antibiotics like Sulfamethoxazole (SMX) pose a significant threat to public health and environmental well-being. To address this issue, effective strategies are being developed to remove antibiotics from the environment. This study investigates the degradation of SMX with a focus on elucidating the mechanism by which extracellular electron transfer (EET) enhances the efficient degradation of the antibiotic. The results show that SMX was significantly degraded (97 %) by Shewanella oneidensis MR-1 after 120 hours in the presence of a bioelectrochemical system (BES) at a concentration of 1 mg L⁻¹, compared to the absence of BES (69 %) at the same concentration and time. BES was observed to simultaneously remove pollutants like SMX while generating electricity at this concentration. Proteomic analysis was further employed to clarify the mechanism behind this process. Three key SMX-degrading proteins; S-ribosylhomocysteine lyase (luxS), Deoxyribose-phosphate aldolase (deoC), and Amidohydrolase which mainly participated in C-S cleavage, S-N hydrolysis and isoxazole ring cleavage were identified. The study demonstrates that S. oneidensis MR-1 can promote the generation of Nicotinamide Adenine Dinucleotide and Adenosine Triphosphate and facilitate electron transfer to enhance the efficient degradation of SMX. The findings of this study provide new insights into the correlation mechanism between SMX degradation and EET, ultimately contributing to innovative solutions for environmental remediation.