Sequential Co-Immobilization of Enzymes on Magnetic Nanoparticles for Efficient l-Xylulose Production.

Multi-enzymatic strategies have shown improvement in bioconversion during cofactor regeneration. In this study, purified l-arabinitol 4-dehydrogenase (LAD) and nicotinamide adenine dinucleotide oxidase (Nox) were immobilized via individual, mixed, and sequential co-immobilization approaches on magnetic nanoparticles, and were evaluated to enhance the conversion of l-arabinitol to l-xylulose. Initially, the immobilization of LAD or Nox on the nanoparticles resulted in a maximum immobilization yield and relative activity of 91.4% and 98.8%, respectively. The immobilized enzymes showed better pH and temperature profiles than the corresponding free enzymes. Furthermore, co-immobilization of these enzymes via mixed and sequential methods resulted in high loadings of 114 and 122 mg/g of support, respectively. Sequential co-immobilization of these enzymes proved more beneficial for higher conversion than mixed co-immobilization because of better retaining Nox residual activity. Sequentially co-immobilized enzymes showed a high relative conversion yield with broader pH, temperature, and storage stability profiles than the controls, along with high reusability. To the best of our knowledge, this is the first report on the mixed or sequential co-immobilization of LAD and Nox on magnetic nanoparticles for l-xylulose production. This finding suggests that selecting a sequential co-immobilization strategy is more beneficial than using individual or mixed co-immobilized enzymes on magnetic nanoparticles for enhancing conversion applications.

Nonsterile Process for Biohydrogen Production: Recent Updates, Challenges, and Opportunities.

Hydrogen (H2), a clean and versatile energy carrier, has recently gained significant attention as a potential solution for reducing carbon emissions and promoting sustainable energy systems. The yield and efficiency of the biological H2 production process primarily depend on sterilization conditions. Various strategies, such as heat inactivation and membrane-based sterilization, have been used to achieve desirable yields via microbial fermentation. Almost every failed biotransformation process is linked to nonsterile conditions at any reaction stage. Therefore, the production of renewable biofuels as alternatives to fossil fuels is more attractive. Pure sugars have been widely documented as a costly feedstock for H2 production under sterile conditions. Biotransformation under nonsterile conditions is more desirable for stable and sustainable operation. Low-cost feeds, such as biowaste, are considered suitable alternatives, but they require appropriate sterilization to overcome the limitations of inherited or contaminating microbes during H2 production. This article describes the status of microbial fermentative processes for H2 production under nonsterile conditions and discusses strategies to improve such processes for sustainable, cleaner production.

Initial experience with 3D laparoscopic choledochal cyst (CDC) excision and hepatico-duodenostomy (HD) in 21 children.

INTRODUCTION: Minimal access surgery has gradually become the standard of care in the management of choledochal cysts (CDC). Laparoscopic management of CDC is a technically challenging procedure that requires advanced intracorporeal suturing skills, and hence, has a steep learning curve. Robotic surgery has the advantages of 3D vision, articulating hand instruments making suturing easy and thus is ideal. However, the non-availability, high costs and necessity for large-size ports are the major limiting factors for robotic procedures in the paediatric population. Use of 3D laparoscopy incorporates the advantage of 3D vision and at the same time allows the use of small-sized conventional laparoscopic instruments. With this background, we discuss our initial experience with the use of 3D laparoscopy using conventional hand instruments in CDC management. AIM: To study our initial experience in the management of CDC in paediatric patients with 3D laparoscopy in terms of feasibility and peri-operative details. MATERIALS AND METHOD: All patients under 12 years of age treated for choledochal cyst in a period of initial 2 years were retrospectively analysed. Demographic parameters, clinical presentation, intra-operative time, blood loss, post-operative events and follow-up were studied. RESULTS: The total number of patients were 21. The mean age was 5.3 years with female preponderance. Abdominal pain was the most common presenting symptom. All patients could be completed laparoscopically. No patient needed conversion to open procedure or re-exploration. The average blood loss was 26.67 ml. None of the patients required a blood transfusion. One patient developed a minor leak postoperatively and was managed conservatively. CONCLUSION: 3D laparoscopic management of CDC in the paediatric age group is safe and feasible. It offers the advantages of depth perception aiding intracorporeal suturing, with the use of small-sized instruments. It is thus a 'bridging the gap' asset between conventional laparoscopy and robotic surgery. LEVEL OF EVIDENCE: Treatment study level IV.

Exploring health coaching and mindfulness as levers for transformation in health: stakeholder perspectives.

BACKGROUND: Health coaching (HC) and mindfulness (MFN) are proven interventions for mobilizing patients' inner resources and are slowly being integrated into public primary care. Since 2015 the medical community in Gibsons BC has integrated physician-led HC and MFN-based programs into team-based care. This exploratory study aimed to understand the mechanisms by which these rural programs helped both patients and clinicians, and to elicit priorities for future study in these fields. METHODS: Using a qualitative participant-engaged constructivist approach in focus groups and large-group graphic facilitation, we elicited perspectives from patients and their physicians during a 1-day event held in September 2018. Thematic analysis of transcripts using Nvivo identified emergent themes that were regularly reviewed with coresearchers, and member checked with participants via online videoconferences held at 6 weeks and 4 months postevent. RESULTS: We identified six main themes relating to the successful implementation of these programs: (i) accessibility and affordability, (ii) offering a toolbox of practical skills, (iii) providing attuned and openhearted care, (iv) generating hope and self-efficacy, (v) experiencing a shared humanity and connection, and (vi) addressing the health of the whole person. CONCLUSION: These themes highlight critical qualities of HC and MFN programs when implemented in a Medicare system. Key features include reducing stigma around mental health through making programs physician-led and a natural part of primary care, enriching accessibility through public funding, and enhancing patient agency through cultivating embodied awareness, self-compassion, and interpersonal skills. These themes inform the next steps to support upscaling these programs to other communities.

Health coaching (HC) and mindfulness (MFN) are proven patient-centered interventions for mobilizing patients' inner resources and changing the trajectories of many chronic physical and psychological conditions. However, though widely available in the private sector, they have been slow to find their way into public primary care in Canada. Since 2015 a rural medical community in BC Canada has integrated physician-led HC and MFN-based programs into primary care. This exploratory study aimed to understand how these programs are working to help both patients and clinicians, providing insights that other communities might use. We elicited perspectives from patient participants and their physicians through focus groups and large-group graphic facilitation during a 1-day event held in September 2018. We identified six themes contributing to successful implementation. Key features include reducing stigma around mental health through making programs publicly accessible and physician-led, and enhancing patient hope and empowerment through cultivating awareness, self-compassion, and interpersonal skills. It also mattered that the facilitators created psychological safety where participants could grow these skills in a nurturing environment, while addressing various dimensions of health. These themes refine our understanding of how these programs can work within public systems to support patient self-management.

An Unusual Case of Type A Posterior Cloaca Associated with 46XX Disorder of Sexual Differentiation with Y Duplication of Urethra.

We report an infant reared as a girl who presented with ambiguous genitalia and urine coming per rectum. On examination, she had minimal clitoromegaly with labial fusion and Y urethral duplication. On investigations, genetic, metabolic, and endocrine causes for disorder of sexual differentiation were ruled out. This girl was operated via a posterior sagittal approach. This case was unique in that the urogenital sinus was deviated posteriorly and opened in the anterior rectal wall and the accessory urethra opened in clitoris, along with ambiguity of external genitalia, thus making it a rare variant of the posterior cloaca. The diagnosis requires a high index of suspicion in clinician and a meticulous examination of the external genitalia. Very little literature is available for this rare anomaly. Few reports have classified them as female pseudohermaphroditism with cloacal and urogenital sinus defects. Hence, we hereby discuss and review the literature for previously reported cases.

Correction to: Biomethanol Production from Methane by Immobilized Co-cultures of Methanotrophs.

[This corrects the article DOI: 10.1007/s12088-020-00883-6.].

Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and Its Potential Application in Bisphenol A Degradation.

In the present study, Rhus vernicifera laccase (RvLac) was immobilized through covalent methods on the magnetic nanoparticles. Fe2O3 and Fe3O4 nanoparticles activated by 3-aminopropyltriethoxysilane followed with glutaraldehyde showed maximum immobilization yields and relative activity up to 81.4 and 84.3% at optimum incubation and pH of 18 h and 5.8, respectively. The maximum RvLac loading of 156 mg/g of support was recorded on Fe2O3 nanoparticles. A higher optimum pH and temperature of 4.0 and 45 °C were noted for immobilized enzyme compared to values of 3.5 and 40 °C for free form, respectively. Immobilized RvLac exhibited better relative activity profiles at various pH and temperature ranges. The immobilized enzyme showed up to 16-fold improvement in the thermal stability, when incubated at 60 °C, and retained up to 82.9% of residual activity after ten cycles of reuses. Immobilized RvLac exhibited up to 1.9-fold higher bisphenol A degradation efficiency potential over free enzyme. Previous reports have demonstrated the immobilization of RvLac on non-magnetic supports. This study has demonstrated that immobilization of RvLac on magnetic nanoparticles is very efficient especially for achieving high loading, better pH and temperature profiles, and thermal- and solvents-stability, high reusability, and higher degradation of bisphenol A.

Posterior Sagittal Approach for Uterovaginal Anastomosis in a Case of Congenital Cervical Atresia with Anorectal Malformation.

Cervical atresia is a rare association with anorectal malformation (ARM) which can be missed till puberty in the presence of normal vaginal orifice. A 12-year-old girl operated for ARM in neonatal age presented with primary amenorrhea. She had a normal vaginal opening, short perineal body, and prolapsed anteposed anus and was diagnosed with cervical agenesis. As the posterior sagittal approach is standard to place the rectum in correct anatomical position, reconstruction of the anus along with adequate perineal body and uterovaginal anastomosis was performed through this approach. This report highlights the utility and versatility of this approach for the management of such complex cases.

Biomethanol Production from Methane by Immobilized Co-cultures of Methanotrophs.

Methanol production by co-culture of methanotrophs Methylocystis bryophila and Methyloferula stellata was examined from methane, a greenhouse gas. Co-culture exhibited higher methanol yield of 4.72 mM at optimum ratio of M. bryophila and M. stellata (3:2) compared to individual cultures. The immobilized co-culture within polyvinyl alcohol (PVA) showed relative efficiency of 90.1% for methanol production at polymer concentration of 10% (v/v). The immobilized co-culture cells within PVA resulted in higher bioprocess stability over free cells at different pH, and temperatures. Free and encapsulated co-cultures showed maximum methanol production of 4.81 and 5.37 mM under optimum conditions, respectively. After five cycles of reusage under batch conditions, free and encapsulated co-cultures retained methanol production efficiency of 23.8 and 61.9%, respectively. The present investigation successfully revealed the useful influence of co-culture on the methanol production over pure culture. Further, encapsulation within the polymeric matrix proved to be a better approach for the enhanced stability of the bioprocess.

Influence of Metal Ions on the Immobilization of ß-Glucosidase Through Protein-Inorganic Hybrids.

Immobilization of enzymes through metal-based system is demonstrated as a promising approach to enhance its properties. In this study, the influence of metals ions, including copper, cobalt and zinc (Zn) on the immobilization of ß-glucosidase (BGL) through the synthesis of protein-inorganic hybrid was evaluated at 4 °C. Among these metal ions-based hybrids, Zn showed the highest encapsulation yield and relative activity of 87.5 and 207%, respectively. Immobilized BGL exhibited higher pH and temperature stability compared to free form. Thermal stability of hybrid improved up to 26-fold at 60 °C. After 10 cycles of reuse, immobilized enzyme retained 93.8% of residual activity. These results suggested that metal ions played a significant role in the enzyme immobilization as a protein-inorganic hybrid. Overall, this strategy can be potentially applied to enhance the properties of enzymes though effective encapsulation for the broad biotechnological applications.

Novel cofactor regeneration-based magnetic metal-organic framework for cascade enzymatic conversion of biomass-derived bioethanol to acetoin.

Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal-organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal-organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.

Trametes versicolor Laccase-Based Magnetic Inorganic-Protein Hybrid Nanobiocatalyst for Efficient Decolorization of Dyes in the Presence of Inhibitors.

In the present investigation, an ecofriendly magnetic inorganic-protein hybrid system-based enzyme immobilization was developed using partially purified laccase from Trametes versicolor (TvLac), Fe3O4 nanoparticles, and manganese (Mn), and was successfully applied for synthetic dye decolorization in the presence of enzyme inhibitors. After the partial purification of crude TvLac, the specific enzyme activity reached 212 U∙mg total protein-1. The synthesized Fe3O4/Mn3(PO4)2-laccase (Fe3O4/Mn-TvLac) and Mn3(PO4)2-laccase (Mn-TvLac) nanoflowers (NFs) exhibited encapsulation yields of 85.5% and 90.3%, respectively, with relative activities of 245% and 260%, respectively, compared with those of free TvLac. One-pot synthesized Fe3O4/Mn-TvLac exhibited significant improvements in catalytic properties and stability compared to those of the free enzyme. Fe3O4/Mn-TvLac retained a significantly higher residual activity of 96.8% over that of Mn-TvLac (47.1%) after 10 reuse cycles. The NFs showed potential for the efficient decolorization of synthetic dyes in the presence of enzyme inhibitors. For up to five reuse cycles, Fe3O4/Mn-TvLac retained a decolorization potential of 81.1% and 86.3% for Coomassie Brilliant Blue R-250 and xylene cyanol, respectively. The synthesized Fe3O4/Mn-TvLac showed a lower acute toxicity towards Vibrio fischeri than pure Fe3O4 nanoparticles did. This is the first report of the one-pot synthesis of biofriendly magnetic protein-inorganic hybrids using partially purified TvLac and Mn.

Encapsulation of Methanotrophs within a Polymeric Matrix Containing Copper- and Iron-Based Nanoparticles to Enhance Methanol Production from a Simulated Biogas.

The production of renewable energy or biochemicals is gaining more attention to minimize the emissions of greenhouse gases such as methane (CH4) and carbon dioxide for sustainable development. In the present study, the influence of copper (Cu)- and iron (Fe)-based nanoparticles (NPs), such as Cu, Fe3O4, and CuFe2O4, was evaluated during the growth of methanotrophs for inoculum preparation and on the development of a polymeric-matrix-based encapsulation system to enhance methanol production from simulated biogas (CH4 and CO2). The use of simulated biogas feed and the presence of NP-derived inoculums produce a remarkable enhancement in methanol production up to 149% and 167% for Methyloferula stellata and Methylocystis bryophila free-cells-based bioconversion, respectively, compared with the use of pure CH4 as a control feed during the growth stage. Furthermore, these methanotrophs encapsulated within a polymeric matrix and NPs-based systems exhibited high methanol production of up to 156%, with a maximum methanol accumulation of 12.8 mmol/L over free cells. Furthermore, after encapsulation, the methanotrophs improved the stability of residual methanol production and retained up to 62.5-fold higher production potential than free cells under repeated batch reusability of 10 cycles. In the presence of CH4 vectors, methanol production by M. bryophila improved up to 16.4 mmol/L and retained 20% higher recycling stability for methanol production in paraffin oil. These findings suggest that Cu and Fe NPs can be beneficially employed with a polymeric matrix to encapsulate methanotrophs and improve methanol production.

Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol.

In this study, a bio-friendly inorganic protein hybrid-based enzyme immobilization system using partially purified Coriolus versicolor laccase (CvLac) was successfully applied to biomass hydrolysis for the enhancement of sugar production aimed at generating biofuels. After four days of incubation, the maximum CvLac production was achieved at 140 U/mg of total protein in the presence of inducers such as copper and wheat bran after four days of incubation. Crude CvLac immobilized through inorganic protein hybrids such as nanoflowers (NFs) using zinc as Zn3(PO4)2/CvLac hybrid NFs (Zn/CvLac-NFs) showed a maximum encapsulation yield of 93.4% and a relative activity of 265% compared to free laccase. The synthesized Zn/CvLac-NFs exhibited significantly improved activity profiles and stability compared to free enzymes. Furthermore, Zn/CvLac-NFs retained a significantly high residual activity of 96.2% after ten reuse cycles. The saccharification of poplar biomass improved ∼2-fold in the presence of Zn/CvLac-NFs, with an 8-fold reduction in total phenolics compared to the control. The Zn/CvLac-NFs treated biomass hydrolysate showed high biological hydrogen (H2) production and ethanol conversion efficiency of up to 2.68 mol/mol of hexose and 79.0% compared to the control values of 1.27 mol of H2/mol of hexose and 58.4%, respectively. The CvLac hybrid NFs are the first time reported for biomass hydrolysis, and a significant enhancement in the production of hydrogen and ethanol was reported. The synthesis of such NFs based on crude forms of diverse enzymes can potentially be extended to a broad range of biotechnological applications.

Synthetic design of methanotroph co-cultures and their immobilization within polymers containing magnetic nanoparticles to enhance methanol production from wheat straw-based biogas.

In this study, various methanotroph co-cultures were designed to enhance methanol production from biogas produced through the anaerobic digestion of wheat straw (WS). Furthermore, whole-cell immobilization was performed using magnetic nanoparticle (MNP)-loaded polymers to develop an efficient bioprocess. The anaerobic digestion of WS by cattle dung yielded 219 L/kg of total solids reduced. Methanol produced was 5.08 and 6.39 mmol/L by pure- and co-cultures from biogas, respectively. The optimization of process parameters enhanced methanol production to 6.82 mmol/L by co-culturing Mithylosinus sporium and Methylocella tundrae. The immobilized co-culture within the MNP-doped polymers exhibited much higher cumulative methanol of up to 70.74 mmol/L than the production of 22.34 mmol/L by free cells after ten cycles of reuse. This study suggests that MNP-doped polymer-based immobilization of methanotrophs is a unique approach for producing renewable fuels from biomass-derived biogas, a greenhouse gas.

Recent Strategies for the Immobilization of Therapeutic Enzymes.

Therapeutic enzymes play important roles in modern medicine due to their high affinity and specificity. However, it is very expensive to use them in clinical medicine because of their low stability and bioavailability. To improve the stability and effectiveness of therapeutic enzymes, immobilization techniques have been employed to enhance the applications of therapeutic enzymes in the past few years. Reported immobilization techniques include entrapment, adsorption, and covalent attachment. In addition, protein engineering is often used to improve enzyme properties; however, all methods present certain advantages and limitations. For carrier-bound immobilization, the delivery and release of the immobilized enzyme depend on the properties of the carrier and enzyme. In this review, we summarize the advantages and challenges of the current strategies developed to deliver therapeutic enzymes and provide a future perspective on the immobilization technologies used for therapeutic enzyme delivery.

Integrating anaerobic digestion of potato peels to methanol production by methanotrophs immobilized on banana leaves.

In the present study, potato peels were subjected to anaerobic digestion (AD) to produce biogas (methane [CH4] and carbon dioxide), which was subsequently used as a substrate for methanol production by methanotrophs. AD resulted in high yields of up to 170 L CH4/kg total solids (TS) from 250 mL substrate (2% TS, w/v). Under optimized conditions, maximum methanol production of 4.97 and 3.36 mmol/L from raw biogas was observed in Methylocella tundrae and Methyloferula stellata, respectively. Immobilization of methanotrophs on banana leaves showed loading of up to 156 mg dry cell mass/g support. M. tundrae immobilized on banana leaves retained 31.6-fold higher methanol production stability, compared to non-immobilized cells. To the best of our knowledge, this is the first study on immobilization of methanotrophs on banana leaves for producing methanol from potato peels AD-derived biogas. Such integrative approaches may be improved through process up-scaling to achieve sustainable development.

Conversion of biogas to methanol by methanotrophs immobilized on chemically modified chitosan.

In this study, chitosan modified with glutaraldehyde (GLA), 3-aminopropyltriethoxysilane (APTES), polyethyleneimine, and APTES followed by GLA (APTES-GLA) as a support material was used to improve methanol production from biogas. Among these support materials, chitosan-APTES-GLA showed the highest increase in immobilization yield and relative efficiency of Methylomicrobium album up to 56.4% and 97.7%, respectively. Maximum cell loading of 236 mg dry cell mass per g-support was observed for M. album., which is 7.7-fold higher than that of chitosan. The immobilized M. album maintained a 23.9-fold higher methanol production compared to free cells after 8 cycles of reuse; it also produced 6.92 mmol·L-1 methanol from biogas that originated from anaerobic digestion of rice straw, thereby validating its industrial application. This is the first report on the immobilization of methanotrophs on chemically modified chitosans to improve cell loading and relative efficiency, and its potential applications in the conversion of greenhouse gases to methanol.

Multidisciplinary Approach for Management of Pediatric Tumors: a Case Series.

Pediatric solid tumors have wide range of presentations. Multidisciplinary approach is often needed for their optimum management. There are no reports discussing such an approach to pediatric tumors involving pediatric surgeons, oncologists and cardiothoracic surgeons together for the management. We report 5 such cases being managed in our institution from 2010 to 2016. All cases needed chemotherapy followed by resection with a team of surgeons involving pediatric surgeon and cardiothoracic surgeon.

Enhanced Saccharification and Fermentation of Rice Straw by Reducing the Concentration of Phenolic Compounds Using an Immobilized Enzyme Cocktail.

The production of bioethanol from rice straw can contribute to the rural economy and provide clean fuel in a sustainable manner. However, phenolic compounds, which are mostly produced during acid pretreatment of biomass, act as inhibitors of fermenting microorganisms. Laccase is well known for its ability to oxidize lignin and phenolic compounds derived from lignocellulosic biomass. In the present study, an immobilized enzyme cocktail containing laccase was isevaluated in regard to its ability to enhance the saccharification and fermentation processes by reducing the amount of phenolic compounds produced. Saccharification of rice straw with the laccase-supplemented immobilized enzyme cocktail reduced phenolic compounds by 73.8%, resulting in a saccharification yield of 84.6%. In addition, improved yeast performance was is noted during the fermentation process, resulting in a 78.3% conversion of sugar into ethanol with an ethanol productivity of 0.478 g/L/h. To the best of our knowledge, this is the first description of the use of an immobilized enzyme cocktail comprised of Celluclast 1.5L, ß-glucosidase, and laccase for the production of bioethanol from rice straw. This study details a potential approach to producing biofuels from agricultural biomass, the applicability of which can be improved through up-scaling.