General Synergistic Hybrid Catalyst Synthesis Method Using a Natural Enzyme Scaffold-Confined Metal Nanocluster.

Due to differences in the chemical properties or optimal reaction conditions of the catalysts, the challenge in the design of bio-chemical hybrid catalysts is that the bio-catalysts or chemical catalysts usually cannot maintain the initial catalytic performance. Herein, we report a general bio-chemical hybrid catalyst synthesis method using a natural enzyme scaffold-confined metal nanocluster. A redox-active enzyme is a nanoreactor that allows access to and reduces metal ions into metal nanoclusters in situ, resulting in the enzyme-confined metal nanocluster hybrid catalyst with a synergistic effect to boost catalytic performance. Specifically, bilirubin oxidase-Ir nanoclusters (BOD-Ir NCs) with catalytic properties for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) are designed. The BOD-Ir NCs exhibit an approximately 2-fold ORR activity compared with pure BOD and a 4-fold OER activity compared with pure Ir NCs. BOD-Ir NCs exhibit stability for over 50,000 s, exceeding that of pure Ir NCs (22,000 s). The synergistic catalytic performance is attributed to the following: the mild preparation condition and matched sizes of BOD and the Ir NCs maintain the natural activity of BOD; the highly conductive Ir NCs improve the ORR activity of BOD; and the confining effect of BOD, which improves the stability and activity of the Ir NCs during the OER. In particular, BOD-Ir NCs exhibit a high half-wave potential of 0.97 V for the ORR and a low overpotential of 319 mV at 10 mA cm-2 for the OER, surpassing most of reported catalysts under neutral conditions. Furthermore, laccase-Ir NCs and glucose oxidase-Pd NCs with synergistic catalytic performances are fabricated, proving the universality of this synthetic method. This facile strategy for designing synergistic hybrid catalysts is expected to be applied to more complex chemical transformations.

Shading minimizes the effects of water deficit in Campomanesia xanthocarpa (Mart) O Berg seedlings / Sombreamento minimiza o efeito do déficit hídrico em mudas de Campomanesia xanthocarpa (Mart) O Berg

The objective of this study was to evaluate the activity of antioxidant enzymes, the functioning of the photosystem II and quality of C. xanthocarpa seedlings cultivated under intermittent water deficit and shading levels and the influence of shading on recovery potential after suspension of the stress conditions. The seedlings were subjected to three levels of shading (0, 30, and 70%), six periods of evaluation (start: 0 days; 1st and 2nd photosynthesis zero: 1st and 2nd P0; 1st and 2nd recovery: 1stand 2nd REC; and END), and two forms of irrigation (control: periodically irrigated to maintain 70% substrate water retention capacity, and intermittent irrigation: suspension of irrigation). The plants subjected to intermittent irrigation conditions at 0% shading showed a reduction in water potential (Ψw) and potential quantum efficiency of photosystem II (Fv/Fm) and maximum efficiency of the photochemical process (Fv/F0) and an increase in basal quantum production of the non-photochemical processes (F0/Fm). Superoxide dismutase (SOD) activity was higher in the leaves than in the roots. The C. xanthocarpa is a species sensitive to water deficit but presents strategies to adapt to an environment under temporary water restriction, which are more temporary are most efficient under shading. The seedlings with water deficit at all levels of shading exhibited higher protective antioxidant activity and lower quality at 0% shading. The shading minimizes prevents permanent damage to the photosystem II and after the re-irrigation, the evaluated characteristics showed recovery with respect to the control group, except POD and SOD activities in the leaves.

O objetivo deste estudo foi avaliar a atividade de enzimas antioxidantes, o funcionamento do fotossistema II e a qualidade de mudas de C. xanthocarpa cultivadas sob déficit hídrico intermitente e níveis de sombreamento e a influência do sombreamento sobre o potencial de recuperação após suspensão das condições de estresse. As mudas foram submetidas a três níveis de sombreamento (0, 30 e 70%), seis períodos de avaliação (início: 0 dias; 1ª e 2ª fotossíntese zero: 1ª e 2ª P0; 1ª e 2ª recuperação: 1ª e 2ª REC; e final), e duas formas de irrigação (controle: periodicamente irrigado para manter 70% da capacidade de retenção de água do substrato, e irrigação intermitente: suspensão da irrigação). As plantas submetidas às condições de irrigação intermitente a 0% de sombreamento apresentaram redução do potencial hídrico (Ψw) e eficiência quântica potencial do fotossistema II (Fv/Fm) e máxima eficiência do processo fotoquímico (Fv/F0) e aumento da produção quantica basal dos processos não fotoquímicos (F0/Fm). A atividade da superóxido dismutase (SOD) foi maior nas folhas do que nas raízes. C. xanthocarpa é uma espécie sensível ao déficit hídrico, mas apresenta estratégias para se adaptar a um ambiente com restrição hídrica temporária, sendo mais eficientes sob sombreamento. As mudas com déficit hídrico em todos os níveis de sombreamento exibiram maior atividade antioxidante protetora e menor qualidade no sombreamento 0%. O sombreamento minimiza danos permanentes ao fotossistema II e após a re-irrigação, as características avaliadas apresentaram recuperação em relação ao grupo controle, exceto atividades de POD e SOD nas folhas.

Process intensification to produce a difficult-to-express therapeutic enzyme by high cell density perfusion or enhanced fed-batch.

Intensified bioprocesses have caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, intensified processes can support high cell densities, higher productivities and longer process times, which together can offer lower cost of goods. In this study two different intensified process modes, high cell density perfusion and enhanced fed-batch, were evaluated and compared with a conventional fed-batch process for a difficult-to-express therapeutic enzyme. The intensified process modes were cultivated with a target cell density of 100 × 106  cells/ml and with alternating tangential flow filtration, ATF, as cell retention device. The processes were designed to resemble an established optimized fed-batch process using the knowledge of this process without new dedicated optimization for the intensified modes. The design strategy included decision of the ratio of feed concentrate to base medium and glucose supplementation, which were based on target cell-specific consumption rates of key amino acids and glucose, using a targeted feeding approach (TAFE). A difficult-to-express therapeutic enzyme with multiple glycosylation sites was expressed and analyzed in the different production processes. The two new intensified processes both achieved 10 times higher volumetric productivity (mg/L/day) with retained protein quality and minor changes to the glycan profile compared to the fed-batch process. The study demonstrates the potential of using intensified processes for sensitive complex enzymes. It is shown here that it is possible to transfer a developed fed-batch process into high cell density processes either in intensified fed-batch or steady-state perfusion without new dedicated optimization. The results demonstrated as well that these intensified modes significantly increase the productivity while maintaining the desired product quality, for instance the same amount of product was obtained in 1 day during the perfusion process than in a whole fed-batch run. Without any prior optimization of the perfusion rate, the high cell density perfusion process resulted in only 1.2 times higher medium cost per gram produced protein.

Overview on Microbial Enzymatic Production of Algal Oligosaccharides for Nutraceutical Applications.

Global requirement for algal foods is increasing, as they are progressively consumed for its nutrition and health. Macroalgae is a proven source of metabolites, proteins, pigments, bioactive compounds, and algal polysaccharides. The unique polysaccharides such as agar, carrageenan, porphyran, alginate, fucoidan, laminarin, and ulvan are known for its wide range of bioactivities and extensively used for applications from tissue engineering to drug delivery. However, there are few limitations due to its high molecular size, low compatibility, and hydrocolloid nature. Hence, the enzymatically produced algal oligosaccharides have drawn tremendous attention due to its green synthesis, solubility, and lower molecular size. They are reported to have bioactivities including antioxidant, antiglycemic, immunostimulatory, anti-inflammatory, and prebiotic activities, which can be used in the healthcare and nutraceutical industry for the manufacture of functional foods and dietary supplements. However, identification of potential microorganisms, producing polysaccharide hydrolyzing enzymes, remains a major bottle neck for efficient utilization of bioactive algal oligosaccharides. This review summarizes the recent developments in the identification and characterization of microbial enzymes for the production of bioactive algal oligosaccharides. This can improve our understanding of bioactive algal oligosaccharides and pave way for efficient utilization of macroalgae to prevent various chronic diseases.

Production of Industrial Enzymes via Pichia pastoris as a Cell Factory in Bioreactor: Current Status and Future Aspects.

Industrial enzymes have been widely preferred in various industries such as chemical production, food & beverage, pharmaceutical, textile, cosmetics, etc. due to the advancements in recent years. They are considered more economic than using whole cells and more environmental-friendly than chemical alternatives. Since the demand for industrial enzymes has been rising, the development of production strategies has been gathered speed. In this respect, the efficiency of Pichia pastoris (P. pastoris) as a host for heterologous protein expression has proved and gained attention due to its great potential for large-scale studies. Especially high-cell density fermentation of P. pastoris is a well-studied and efficient method. Moreover, the improvements in the state of art gene-editing tools have broadened the possibilities of strain improvement for P. pastoris. This review summarized the role of P. pastoris as a cell factory by accentuating the accomplishments in biocatalyst production. Moreover, the benefits and challenges of the most relevant expression systems named Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), P. pastoris and recent evolvements and future directions were revealed in detail. Subsequently, offers for prospects and the latest evolvements to enhance the recombinant protein production were discussed.

Environmental Assessment of Enzyme Production and Purification.

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme-1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.

Industrial Relevance of Trichoderma reesei as an Enzyme Producer.

Trichoderma reesei's potential as a rapid and efficient biomass degrader was first recognized in the 1950s when it was isolated from Army textiles during World War II. The microbe secreted cellulases that were degrading cotton-based tents and clothing of service members stationed on the Solomon Islands. In the 1970s, at the time of the first global oil crisis, research interest in T. reesei gained popularity as it was explored as part of the solution to the worlds growing dependence on fossil fuels. Much of this early work focused on classical mutagenesis and selection of hypercellulolytic strains. This early lineage was used as a starting point for both academic research with the goal of understanding secretion and regulation of expression of the complex mixture of enzymes required for cellulosic biomass decay as well as for its development as a host for industrial enzyme production. In 2001, at the onset of the second major oil crisis, the US Department of Energy supported research programs in microbial cellulases to produce ethanol from biomass which led to another surge in the study of T. reesei. This further accelerated the development of molecular biology and recombinant DNA tools in T. reesei. In addition to T. reesei's role in bio-ethanol production, it is used to produce industrial enzymes with a broad range of applications supporting the bio-based economy. To date there are around 243 commercially available enzyme products manufactured by fermentation of microorganisms; 30 of these are made using Trichoderma as a host, 21 of which are recombinant products sold for use in food, feed, and technical applications including textiles and pulp and paper.

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine.

Due to its clear inherited backgrounds as well as simple and diverse genetic manipulation systems, Bacillus subtilis is the key Gram-positive model bacterium for studies on physiology and metabolism. Furthermore, due to its highly efficient protein secretion system and adaptable metabolism, it has been widely used as a cell factory for microbial production of chemicals, enzymes, and antimicrobial materials for industry, agriculture, and medicine. In this mini-review, we first summarize the basic genetic manipulation tools and expression systems for this bacterium, including traditional methods and novel engineering systems. Secondly, we briefly introduce its applications in the production of chemicals and enzymes, and summarize its advantages, mainly focusing on some noteworthy products and recent progress in the engineering of B. subtilis. Finally, this review also covers applications such as microbial additives and antimicrobials, as well as biofilm systems and spore formation. We hope to provide an overview for novice researchers in this area, offering them a better understanding of B. subtilis and its applications.

Termitomyces heimii Associated with Fungus-Growing Termite Produces Volatile Organic Compounds (VOCs) and Lignocellulose-Degrading Enzymes.

Termitomyces fungi associated with fungus-growing termites are the edible mushrooms and can produce useful chemicals, enzymes, and volatile organic compounds (VOCs) that have both fuel and biological potentials. To this purpose, we examined the Termitomyces mycelial growth performance on various substrates, clarified lignocellulose-degrading enzyme activity, and also identified the VOCs produced by Termitomyces. Our results indicated that the optimal nutrition and condition requirements for mycelial growth are D-sorbitol, D-(+)-glucose, and D-(-)-fructose as carbon sources; peptone as well as yeast extract and ammonium tartrate as nitrogen sources; and Mn2+, Na+, and Mg2+ as metal ions with pH range from 7.0 to 8.0. Besides, the orthogonal matrix method results revealed that the ideal composition for mycelial growth is 20 g/L D-(-)-fructose, 5 g/L yeast extract, 0.5 g/L Mg2+, and pH = 7. We also screened various substrates composition for the activity of lignocellulose-degrading enzymes, i.e., lignin peroxidase, manganese peroxidase, ß-glucosidase, a-L-arabinofuranosidase, and laccase. Furthermore, we identified 37 VOCs using GC-MS, and the most striking aspect was the presence of a big series of alcohols and acids, collectively constituted about 49% of the total VOCs. Ergosta-5, 8, 22-trien-3-ol, (3.beta.,22E) was the most plenteous compound constituted 30.369%. This study hopes to establish a better understanding for researchers regarding Termitomyces heimii cultivation on a large scale for the production of lignocellulosic enzymes and some fungal medicine.

Proteomic analysis on Aspergillus strains that are useful for industrial enzyme production.

A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-proteins, likely in signal transduction, and other proteins were also investigated. Abbreviations: 2-D: two-dimensional; UPR: unfolded protein response; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; PDI: protein disulfide-isomerase; ROS: reactive oxygen species; RESS: Repression under Secretion Stress; CSAP: Conserved Small Abundant Protein; TCTP: translationally controlled tumor protein.

Structure, function, and biosynthesis of nickel-dependent enzymes.

Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni-superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]-carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase and [NiFe]-hydrogenase, and even more complex cofactors in methyl-CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo-enzymes.

Construction and characterization of a chimeric lysin ClyV with improved bactericidal activity against Streptococcus agalactiae in vitro and in vivo.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.

Report on biopharmaceutical profile of recent biotherapeutics and insilco docking studies on target bindings of known aptamer biotherapeutics.

Accumulated Toxicity, disease recurrence and drug resistivity problems have been observed due to the synthetic and semisynthetic therapeutic practices, which alternatively led to focus on Bio-therapeutics production than xenobiotics. Quick plasma clearance and high potency are the reasons for trending research with huge pharma market of numerous Bio-therapeutics than ever before. Researchers proved that most of the nano and micro Bio-therapeutics have multiple beneficial therapeutic effects. We have analyzed the past, and present scenario of some notable clinically approved Bio-therapeutics to identify the future formulation needs with advanced techniques. Protein-related drugs are the foremost Bio-therapeutics such as antibodies, enzymes, and short, fragmented polypeptides show aggregation properties during storage, naked peptide moieties are resisted by the polar cell membrane, and also the antidrug antibodies were reported. Even though Nucleic acid nano-bodies are excellent target binders than proteins, they had only a few minutes of half-life. Maintaining homogeneousness upon storage of Bio-therapeutics is still a significant challenge in industrial-scale formulation. Notably, plant systems are identified as most useful cost-effective hosts to produce human enzymes than animal systems without any possible viral loads. Irrespective of numerous advancements in routes of administration and additives, subcutaneous is still a golden one to achieve better dynamics. Additionally, the interactions and effective bonds made by each class of well-known aptamer biotherapeutics which are considered as future drugs were studied.

CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.

Zearalenone Induces Endoplasmic Reticulum Stress and Modulates the Expression of Phase I/II Enzymes in Human Liver Cells.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species; however, its mechanisms of action in human livers have not been fully elucidated. Thus, we investigated the toxic mechanisms of ZEN in human liver cells. HepG2 cells were treated with ZEN (0-40 µg/mL) for up to 24 h. A significant decrease in cell viability was observed after treatment with 20 and 40 µg/mL of ZEN, including a significant increase in apoptosis and reactive oxygen species production. ZEN increased GRP78 and CHOP, and eIF2α phosphorylation, indicating ER stress; elevated transcription of the autophagy-associated genes, beclin1 and LC3, and translation of LC3; and increased phase I metabolism by increasing PXR and CYP3A4. The protein expression level of CYP3A4 was higher with ZEN treatment up to 20 µg/mL, but remained at the control level after treatment with 40 µg/mL ZEN. In phase II metabolism, Nrf2 activation and UGT1A expression were increased with ZEN treatment up to 20 µg/mL. Treating cells with an ER stress inhibitor alleviated ZEN-induced cell death and autophagy, and inhibited the expression of phase I/II enzymes. Overall, high ZEN concentrations can modulate the expression of phase I/II enzymes via ER stress and reduced protein levels in human liver cells.

Light-regulated synthesis of extra- and intracellular enzymes related to wood degradation by the white rot fungus Cerrena unicolor during solid-state fermentation on ash sawdust-based medium.

The light-dependent metabolism of the white rot basidiomycete Cerrena unicolor FCL139 has already been demonstrated using transcriptomic and Biolog-based approaches. To further analyze the influence of light on C. unicolor wood degradation, we measured the activity of an array of CAZymes (carbohydrate-active enzymes) and enzymes involved in the redox system of fungal cells associated with lignolysis. Extra- and intracellular enzymatic extracts were obtained from solid-state ash sawdust C. unicolor cultures cultivated for 14 days under red, blue, green, or white light conditions, or in the dark. Light greatly influenced the synthesis of MnP, total cellulases, endo-1,4-ß-glucanase, endo-1,4-ß-xylanase, catalase, and superoxide dismutase. The production of MnP and catalase was evidently stimulated by white light. It is also worth noticing that blue light caused a gradual increase in the activity of total cellulases throughout the entire period of C. unicolor growth. Moreover, endo-1,4-ß-glucanase showed the highest activity on day 13 of fungus cultivation and the production of laccase and ß-glucosidase appeared to be the least influenced by light. However, the strongest activity of the endo-1,4-ß-xylanase was observed in the dark. It seemed that light not only influenced the regulation of the synthesis of the wood-degrading enzymes at different levels, but also acted indirectly by affecting production of enzymes managing harmful lignin by-products causing oxidative stress. The ability of the fungus to decompose woody plant material is clearly influenced by environmental factors.

Production and Purification of Therapeutic Enzymes.

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.

Production of Therapeutic Enzymes by Lentivirus Transgenesis.

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.

Quality Control and Downstream Processing of Therapeutic Enzymes.

Therapeutic enzymes are a commercially minor but clinically important area of biopharmaceuticals. An array of therapeutic enzymes has been developed for a variety of human diseases, including leukaemia and enzyme-deficiency diseases such as Gaucher's disease. Production and testing of therapeutic enzymes is strictly governed by regulatory bodies in each country around the world, and batch-to-batch consistency is crucially important. Manufacture of a batch starts with the fermentation or cell culture stage. After expression of the therapeutic enzyme in a cell culture bioreactor, robust and reproducible protein purification, or downstream processing (DSP) of the target product, is critical to ensuring safe delivery of these medicines. Modern processing technology, including the use of disposable processing equipment, has greatly improved the DSP development pathway in terms of robustness and speed to clinic. Once purified, the drug substance undergoes rigorous quality control (QC) testing according to current regulatory guidance, to enable release to the clinic and patient. QC testing is conducted to ensure the safety, purity, identity, potency and strength of the medicinal product, requiring multiple analytical methods that are rigorously validated and monitored for robust performance. Several case studies, including L-asparaginase and asfotase alfa, are discussed to illustrate the methods described herein.

A novel and cost-effective methodology for enhanced production of industrially valuable alkaline xylano-pectinolytic enzymes cocktail in short solid-state fermentation cycle.

The aim of this study was to enhance the production of xylano-pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro-residues extract-based inoculum on yield and fermentation time of xylano-pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro-based residues. Enzymes production parameters were optimized through two-stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K2 HPO4 0.22%, MgSO4 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro-based crude xylan in production media 0.45%, and agro-based crude xylan-pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano-pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro-residues. The activity of different types of pectinase enzymes such as exo-polymethylgalacturonase (exo-PMG), endo-PMG, exo-polygalacturonase (exo-PG), endo-PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano-pectinolytic enzymes in short solid-state fermentation cycle using agro-residues extract-based inoculum and production media.