High Molecular Weight Substrates Research Articles

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C9H8O3) compared to virtually no PHB titer from acetate (C2H3NaO2). The maximum yield did not improve further when grown on coniferyl alcohol (C10H12O3), but comparison of the PHB profiles showed that coniferyl alcohol's higher carbon content resulted in a higher rate of PHB production. Combined experimental results revealed that cytoplasmic space may be a limiting factor for maximum PHB titer. In order to obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. The model yielded several engineering design strategies including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction (phaA). Based on these strategies, utilization of butyrate was predicted and subsequently validated to produce PHB. Model analysis also explained why nitrogen starvation was not essential for PHB production and revealed that renewable and abundant lignin aromatics are ideal candidates for PHB production. Most importantly, the generality of the derived design rules allows them to be applied to any PHB-producing microbe with similar metabolic features.

New thermostable endoglucanase from Spirochaeta thermophila and its mutants with altered substrate preferences.

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates-soluble oligosaccharides. This issue was addressed in the current study using enzyme engineering and an enzyme evolution approach. To this end, a hypothetical endoglucanase from a thermostable bacterium Spirochaeta thermophila was for the first time cloned and characterized. The wild-type enzyme was used as a starting point for mutagenesis and molecular evolution toward a preference for the higher molecular weight substrates. The best of the evolved enzymes was more active than the wild-type enzyme toward high molecular weight substrate at temperatures below 45 °C (3-fold more active at 30 °C) and showed little or no activity with low molecular weight substrates. These findings can be instrumental in bioeconomy sectors, such as second-generation biofuels and biomaterials from lignocellulosic biomass. KEY POINTS: • A new thermostable endoglucanase was characterized. • The substrate specificity of this endoglucanase was changed by means of genetic engineering. • A mutant with a preference for long molecular weight substrate was obtained and proposed to be beneficial for cellulose fiber modification.

Bacteria mediated Fenton-like reaction drives the biotransformation of carbon nanomaterials

Carbon nanomaterials (CNs), which gain heightened attention as novel materials, are increasingly incorporated into daily products and thus are released into the environment. Limited research on CNs environmental fates lags their industry growth, only few bacteria have been confirmed to biotransform CNs and the mechanism behind has not been revealed yet. In this study, four types of commercial CNs, i.e. graphene oxide (GO), reduced graphene oxide (RGO), single walled carbon nanotubes (SWCNTs), and oxidized (carboxylated) SWCNTs, were selected for investigation. The biotransformation of CNs by Labrys sp. WJW, which could grow with these CNs as the sole carbon source, was investigated. The bacterial transformation was proved by qPCR, transmission electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, liquid chromatography/time-of-flight/mass spectrometry, and gas chromatograph-mass spectrometry analyses. The biotransformation resulted in morphology change, defect increase and functional group change of these CNs. Furthermore, the underlying mechanism of CNs biodegradation mediated by extracellular Fenton-like reaction was demonstrated. In this reaction, the OH production was mediated by reduction of H2O2 involved a continuous cycle of Fe(II)/Fe(III). These findings reveal a novel degradation mechanism of microorganism towards high molecular weight substrate, which will provide a new insight into the environmental fate of CNs and the guidance for their safer use.

Improved development in magnetic Xyl-CLEAs technology for biotransformation of agro-industrial by-products through the use of a novel macromolecular cross-linker

Cross-Linked Enzyme Aggregates (CLEAs) technologies for enzyme immobilization are influenced by mass transference problems as the degree of molecular crosslinking achieved strongly affects the enzyme exposure to the substrates. Therefore, this work seeks to improve the accessibility of high molecular weight substrates by using macromolecular cross-linkers to the synthesis of a xylanolytic biocatalyst. After confirming that commercial polymers used as macromolecular cross-linkers significantly upgraded the xylanase activity from a crude preparation, a novel biopolymer/amyloid protein complex (BPAP) extracted from a microbial biofilm was used producing a remarkable recovery (83%) of the enzyme activity.A response surface methodology was applied to contrast the features of a previously developed biocatalyst with glutaraldehyde (GA@Xyl-CLEAs) and a novel one synthesized with BPAP combined with functionalized magnetic nanoparticles: mBPAP@Xyl-CLEAs. It was observed that the crosslinking agent used was the factor that most affected the enzyme activity. Also, the mBPAP system showed a similar and higher hydrolytic activity than those synthesized with GA, which was not affected by the mNPs/protein ratio. Finally, the mBPAP@Xyl-CLEAs were successfully tested for xylooligosaccharides production from agroindustrial-derived substrates, making this technology a promising practice to obtain green and suitable biocatalysts.

Candidatus Abditibacter, a novel genus within the Cryomorphaceae, thriving in the North Sea

Coastal phytoplankton blooms are frequently followed by successive blooms of heterotrophic bacterial clades. The class Flavobacteriia within the Bacteroidetes has been shown to play an important role in the degradation of high molecular weight substrates that become available in the later stages of such blooms. One of the flavobacterial clades repeatedly observed over the course of several years during phytoplankton blooms off the coast of Helgoland, North Sea, is Vis6. This genus-level clade belongs to the family Cryomorphaceae and has been resistant to cultivation to date. Based on metagenome assembled genomes, comparative 16S rRNA gene sequence analyses and fluorescence in situ hybridization, we here propose a novel candidate genus Abditibacter, comprising three novel species Candidatus Abditibacter vernus, Candidatus Abditibacter forsetii and Candidatus Abditibacter autumni. While the small genomes of the three novel photoheterotrophic species encode highly similar gene repertoires, including genes for degradation of proteins and algal storage polysaccharides such as laminarin, two of them – Ca. A. vernus and Ca. A. forsetii – seem to have a preference for spring blooms, while Ca. A. autumni almost exclusively occurs in late summer and autumn.

Properties of a Non-canonical Complex Formed Between a Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and α-Chymotrypsin.

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.

Selfish, sharing and scavenging bacteria in the Atlantic Ocean: a biogeographical study of bacterial substrate utilisation

Identifying the roles played by individual heterotrophic bacteria in the degradation of high molecular weight (HMW) substrates is critical to understanding the constraints on carbon cycling in the ocean. At five sites in the Atlantic Ocean, we investigated the processing of organic matter by tracking changes in microbial community composition as HMW polysaccharides were enzymatically hydrolysed over time. During this investigation, we discovered that a considerable fraction of heterotrophic bacteria uses a newly-identified ‘selfish’ mode of substrate processing. We therefore additionally examined the balance of individual substrate utilisation mechanisms at different locations by linking individual microorganisms to distinct substrate utilisation mechanisms. Through FISH and uptake of fluorescently-labelled polysaccharides, ‘selfish’ organisms were identified as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. ‘Sharing’ (extracellular enzyme producing) and ‘scavenging’ (non-enzyme producing) organisms predominantly belonged to the Alteromonadaceae and SAR11 clades, respectively. The extent to which individual mechanisms prevail depended on the initial population structure of the bacterial community at a given location and time, as well as the growth rate of specific bacteria. Furthermore, the same substrate was processed in different ways by different members of a pelagic microbial community, pointing to significant follow-on effects for carbon cycling.

Prokaryotic assemblages within permafrost active layer at Edmonson Point (Northern Victoria Land, Antarctica)

This study was aimed at gaining insights on the prokaryotic community (in terms of both taxonomic composition and activities) inhabiting the active layer at Edmonson Point, an ice-free area on the eastern slope at the foot of Mount Melbourne (Northern Victoria Land, Antarctica). Samples were collected during the thawing period, when microbial physiological activities are restored to utilize previously frozen organic substrates. Despite the very small cell sizes (<0.1 μm3), indicating the occurrence of stressed, dormant and/or starved cells, the prokaryotic communities appeared to be metabolically active in the decomposition of high molecular weight (>600 Da) substrates, as indicated also by the obtained rates of enzymatic hydrolytic activities over proteolytic, glycolitic and phosphoric compounds. Taxonomical composition showed that Proteobacteria, Actinobacteria and Firmicutes dominated the prokaryotic community, with most of their members playing crucial roles in organic matter turnover, as well as nitrogen cycling, or entering a viable but not cultivable state to cope with continuously changing environmental conditions, such as in the case of the active layer. Finally, non-autochthonous bacteria (mainly of marine origin) were detected and they probably contribute to the organic matter turnover within such cold terrestrial habitat.This research provides the first comprehensive account of the prokaryotic communities inhabiting the Antarctic permafrost and contributes to existing information on the response of their abundance and metabolism in a permafrost area that undergoes to seasonal changes (e.g. in terms of temperature, water availability and ice presence).

Characterization of a topologically unique oxygenase from Sphingobium sp. PNB capable of catalyzing a broad spectrum of aromatics.

A Rieske non-heme iron ring-hydroxylating oxygenase (RHO) from Sphingobium sp. PNB involved in the initial oxidation of a wide range of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs) was investigated. The RHO was shown to comprise of the gene products of distantly located ahdA1f-ahdA2f, ahdA3 and ahdA4 genes, which encoded the oxygenase α- and β-subunits, ferredoxin and reductase, respectively. In silico structural analysis of AhdA1f revealed a very large substrate-binding pocket, satisfying the spatial requirements to accommodate high molecular weight substrates. In addition, an atypical substrate access channel was noticed from the topology analysis of the oxygenase. Guided by molecular docking studies, dioxygenation of several PAHs as well as alkyl- and aryl benzenes was examined with the recombinant AhdA1fA2f expressed in Escherichia coli. Chromatographic and mass spectrometric analyses confirmed that AhdA1fA2f displays broad substrate specificity towards a wide range of aromatic hydrocarbons including potential xenobiotics, demonstrating metabolic robustness of strain PNB.

Synthesis and Characterization of Highly Stabilized Polymer–Trypsin Conjugates with Autolysis Resistance

Protein digestion by trypsin has been widely used in many industrial and research applications. However, extensive use of trypsin is limited because of the rapid decrease in enzymatic activity caused by autolysis at optimal pH and temperature. To improve the enzymatic performance of trypsin, we synthesized highly stabilized polymer–trypsin conjugates using vinylmethylether-maleic acid copolymer (VEMAC) via multi-point attachment. The VEMAC modification significantly enhanced the thermal stability of trypsin, and the resulting conjugates showed a strong resistance to autolysis. VEMAC-modified trypsin (VEMAC-Tryp) showed maximum activity at 55 °C and at 1.4-fold higher levels than that of unmodified trypsin. Bovine serum albumin was effectively digested by VEMAC-Tryp, indicating that the modified trypsin can be used for digestion of high molecular weight substrates. VEMAC modification is a simple and cost-effective strategy to obtain fully active modified enzymes, and may be used to develop bioreactors.

Construction and characterization of protein-encapsulated electrospun fibermats prepared from a silica/poly(?- glutamate) hybrid

Event Abstract Back to Event Construction and characterization of protein-encapsulated electrospun fibermats prepared from a silica/poly(γ- glutamate) hybrid Toshihisa Mizuno1*, Akiko Obata1*, Shuhei Koeda1, Makoto Shimada1*, Koji Mizuno1*, Makito Iguchi1*, Julian Jones2* and Toshihiro Kasuga1* 1 Nagoya Institute of Technology, Graduate School of Engineering, Japan 2 Imperial College London, Faculty of Engineering, Department of Materials, United Kingdom Protein-encapsulated fibermats are an attractive platform for protein-based bioactive materials. But the choice of methods is still limited and the previously described methods are not broadly amenable to a wide-range of proteins. In this study, therefore we studied new polymeric material for constructing protein-encapsulated fibermats, in which protein molecules are encapsulated within the nanofibers of fibermats without causing deleterious changes to protein structure or function. We constructed a protein-encapsulated fibermat using the poly(g-glutamate) (PGA)/(3-glycidyloxypropyl)-trimethoxysilane (GPTMS) hybrid as a precursor for electrospinning. Since the PGA/GPTMS hybrid is water soluble, protein molecules can be added to the precursor in an aqueous solution, significantly enhancing protein stability. Polycondensation during electrospinning (in-flight polycondensation) makes the obtained fibermats water-insoluble, which stabilizes the fibermat structure so that it is resistant to degradation in aqueous buffer. The molecular structure of the PGA/GPTMS hybrid gives rise to unique molecular permeability, which alters the selectivity and specificity of biochemical reactions involving the encapsulated enzymes; lower molecular-weight (MW) substrates can permeate the nanofibers, promoting enzyme activity, but higher MW substrates such as inhibitor peptides cannot permeate the nanofibers, suppressing enzyme activity. We present an effective method of encapsulating bioactive molecules while maintaining their structure and function, increasing the versatility of electrospun fibermats for constructing various bioactive materials. The present work was carried out under the framework of Academic Unit Cooperation Program between Frontier Research Institute for Materials Science, Nagoya Institute of Technology and Department of Materials, Imperial College London. Keywords: nanofiber, protein, Biofunction Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Nano-structured materials for unique functions Citation: Mizuno T, Obata A, Koeda S, Shimada M, Mizuno K, Iguchi M, Jones J and Kasuga T (2016). Construction and characterization of protein-encapsulated electrospun fibermats prepared from a silica/poly(γ- glutamate) hybrid. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01546 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Toshihisa Mizuno, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, Email1 Dr. Akiko Obata, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, obata.akiko@nitech.ac.jp Dr. Makoto Shimada, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, m.shimada.853@nitech.jp Dr. Koji Mizuno, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, 24111144@stn.nitech.ac.jp Dr. Makito Iguchi, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, cjh12008@nitech.jp Dr. Julian Jones, Imperial College London, Faculty of Engineering, Department of Materials, London, United Kingdom, julian.r.jones@imperial.ac.uk Dr. Toshihiro Kasuga, Nagoya Institute of Technology, Graduate School of Engineering, Nagoya, Japan, kasuga.toshihiro@nitech.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Toshihisa Mizuno Akiko Obata Shuhei Koeda Makoto Shimada Koji Mizuno Makito Iguchi Julian Jones Toshihiro Kasuga Google Toshihisa Mizuno Akiko Obata Shuhei Koeda Makoto Shimada Koji Mizuno Makito Iguchi Julian Jones Toshihiro Kasuga Google Scholar Toshihisa Mizuno Akiko Obata Shuhei Koeda Makoto Shimada Koji Mizuno Makito Iguchi Julian Jones Toshihiro Kasuga PubMed Toshihisa Mizuno Akiko Obata Shuhei Koeda Makoto Shimada Koji Mizuno Makito Iguchi Julian Jones Toshihiro Kasuga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Construction and Characterization of Protein-Encapsulated Electrospun Fibermats Prepared from a Silica/Poly(γ-glutamate) Hybrid

Protein-encapsulated fibermats are an attractive platform for protein-based bioactive materials. However, the choice of methods is still limited and not applicable to a wide range of proteins. In this study, we studied new polymeric materials for constructing protein-encapsulated fibermats, in which protein molecules are encapsulated within the nanofibers of fibermats without causing deleterious changes to protein structure or function. We constructed a protein-encapsulated fibermat using the poly(γ-glutamate) (PGA)/(3-glycidyloxypropyl)-trimethoxysilane (GPTMS) hybrid as a precursor for electrospinning. Because the PGA/GPTMS hybrid is water-soluble, protein molecules can be added to the precursor in an aqueous solution, significantly enhancing protein stability. Polycondensation during electrospinning (in-flight polycondensation) makes the obtained fibermats water-insoluble, which stabilizes the fibermat structure such that it is resistant to degradation in aqueous buffer. The molecular structure of the PGA/GPTMS hybrid gives rise to unique molecular permeability, which alters the selectivity and specificity of biochemical reactions involving the encapsulated enzymes; lower molecular-weight (MW) substrates can permeate the nanofibers, promoting enzyme activity, but higher MW substrates such as inhibitor peptides cannot permeate the nanofibers, suppressing enzyme activity. We present an effective method of encapsulating bioactive molecules while maintaining their structure and function, increasing the versatility of electrospun fibermats for constructing various bioactive materials.

Truncated Variants of Serratia proteamaculans Oligopeptidase B Having Different Activities.

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

Biofilm increases permeate quality by organic carbon degradation in low pressure ultrafiltration

We investigated the influence of biofouling of ultrafiltration membranes on the removal of organic model foulants and ultimately on the quality of permeate. Gravity Driven Membrane ultrafiltration (GDM) membrane systems were operated with modified river water during five weeks without control of the biofilm formation. Three GDM systems were studied: two systems with biofilms exposed to (A) variable or (B) constant load of organic foulants, and (C) one system operated without biofilm and exposed to constant foulant loading. Biodegradable dextran or non-biodegradable polystyrene sulfonate model foulants were tested. Substrate biodegradability was confirmed by Size Exclusion Chromatography (SEC) and by degradation batch tests (D). The GDM systems (A) and (B) were fed with pre-filtered river water supplemented with dextran (Dex) of 1, 150 or 2000 kDa, or polystyrene sulfonate (PSS) of 1 or 80 kDa at concentrations of 2–3.5 mgC L−1. In exp. (C) the feed water consisted of deionized water with 25 mgC L−1 of either PSS 1, 80 kDa or Dex 2000 kDa.The biofilm formation on UF membrane surfaces controlled the foulant permeation and thus the permeate quality. Biofilms exposed to continuous foulant loading (exp. B) degraded low molecular weight (LMW) biodegradable foulants (1 kDa Dex), which improved the permeate quality. For high molecular weight (HMW) substrates (150, 2000 kDa Dex), the improvement of the permeate quality was observed after 7 days of biofilm formation, and resulted from the foulant hydrolysis followed by degradation. For non-biodegradable foulants, an improvement of 20% of the retention was observed for the polystyrene (1, 80 kDa PSS) due to the presence of biofilms on membrane surfaces. For variable foulant loading (exp. A) the biofilms hydrolysed the large biodegradable foulants but did not degraded them fully, which resulted a deterioration of the permeate quality (except for the LMW dextran (1 kDa) that was fully degraded). Overall, the “biofilm + membrane” composite retained a larger amount of biodegradable foulant than the membrane alone, due to the activity of the biofilm. However, this resulted in an increased biofilm accumulation and reduced flux. In presence of the biofilm, the highest fluxes were observed for control (no foulant) and for small non-biodegradable foulants (PSS 1 kDa). Low fluxes were observed for the accumulating on membrane surface or degradable foulants (exp. B). But, the lowest fluxes were observed in absence of the biofilm (exp. C) due to physical accumulation of the foulants (PSS 80 kDa and Dextran 2000 kDa). Overall our study demonstrates that the presence of biofilms on membrane surfaces has some benefits: (i) biofilm helps to increase the permeate quality and (ii) biofilms protect the membrane from further fouling. Permeate flux stabilizes in the case of biofilm-membrane composite, while it continuously declines in the case of the membrane only.

Immobilization of β-galactosidase from Lactobacillus plantarum HF571129 on ZnO nanoparticles: characterization and lactose hydrolysis.

β-Galactosidase from Lactobacillus plantarum HF571129 was immobilized on zinc oxide nanoparticles (ZnO NPs) using adsorption and cross-linking technique. Immobilized β-galactosidase showed broad-spectrum pH optima at pH 5-7.5 and temperature 50-60°C. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that β-galactosidase successfully immobilized onto supports. Due to the limited diffusion of high molecular weight substrate, K m of immobilized enzyme slightly increased from 6.64 to 10.22mM, while V max increased from 147.5 to 192.4µmolmin(-1)mg(-1) as compared to the soluble enzyme. The cross-linked adsorbed enzyme retained 90% activity after 1-month storage, while the native enzyme showed only 74% activity under similar incubation conditions. The cross-linked β-galactosidase showed activity until the seventh cycle and maintained 88.02% activity even after the third cycle. The activation energy of thermal deactivation from immobilized biocatalyst was 24.33kcal/mol with a half-life of 130.78min at 35°C. The rate of lactose hydrolysis for batch and packed bed was found to be 0.023 and 0.04min(-1).

Ecophysiology of uncultivated marine euryarchaea is linked to particulate organic matter.

Particles in aquatic environments host distinct communities of microbes, yet the evolution of particle-specialized taxa and the extent to which specialized microbial metabolism is associated with particles is largely unexplored. Here, we investigate the hypothesis that a widely distributed and uncultivated microbial group—the marine group II euryarchaea (MGII)—interacts with living and detrital particulate organic matter (POM) in the euphotic zone of the central California Current System. Using fluorescent in situ hybridization, we verified the association of euryarchaea with POM. We further quantified the abundance and distribution of MGII 16 S ribosomal RNA genes in size-fractionated seawater samples and compared MGII functional capacity in metagenomes from the same fractions. The abundance of MGII in free-living and >3 μm fractions decreased with increasing distance from the coast, whereas MGII abundance in the 0.8–3 μm fraction remained constant. At several offshore sites, MGII abundance was highest in particle fractions, indicating that particle-attached MGII can outnumber free-living MGII under oligotrophic conditions. Compared with free-living MGII, the genome content of MGII in particle-associated fractions exhibits an increased capacity for surface adhesion, transcriptional regulation and catabolism of high molecular weight substrates. Moreover, MGII populations in POM fractions are phylogenetically distinct from and more diverse than free-living MGII. Eukaryotic phytoplankton additions stimulated MGII growth in bottle incubations, providing the first MGII net growth rate measurements. These ranged from 0.47 to 0.54 d−1. However, MGII were not recovered in whole-genome amplifications of flow-sorted picoeukaryotic phytoplankton and heterotrophic nanoflagellates, suggesting that MGII in particle fractions are not physically attached to living POM. Collectively, our results support a linkage between MGII ecophysiology and POM, implying that marine archaea have a role in elemental cycling through interactions with particles.

Identification of a novel, nanobody‐induced, mechanism of TAFI inactivation and its in vivo application

Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates. We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg⁻¹) to 4.4 U mg⁻¹ (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37 °C. Incubation of mTAFI with VHH-mTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice. The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50°C and reaction time from 5 to 10minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, Km of immobilized enzyme slightly increased from 1.017 to 1.055mgml−1, while Vmax decreased from 23,800 to 19,392μMmin−1 as compared to free enzyme. After 120h entrapped pectinase retained their activity up to 82% and 71% at 30°C and 40°C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle.

Dimerization mediates thermo-adaptation, substrate affinity and transglycosylation in a highly thermostable maltogenic amylase of Geobacillus thermoleovorans.

BackgroundMaltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical α-amylases. Several bacterial malto-genic amylases with temperature optima of 40–60°C have been previously characterized. The thermo-adaption, substrate preferences and transglycosylation aspects of extremely thermostable bacterial maltogenic amylases have not yet been reported.Methodology/Principal FindingsThe recombinant monomeric and dimeric forms of maltogenic α-amylase (Gt-Mamy) of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80°C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90°C and mediates its enthalpy-driven conformational thermostabilization. Furthermore, dime-rization regulates preferential substrate binding of the enzyme. The substrate preference switching of Gt-Mamy upon dimerization has been confirmed from the substrate-binding affinities of the enzyme for various high and low molecular weight substrates. There is an alteration in Km and substrate hydrolysis efficiency (Vmax/Km) of the enzyme (for cyclodex-trins/starch) upon dimerization. N-terminal truncation indicated the role of N-terminal 128 amino acids in the thermostabilization and modulation of substrate-binding affinity. This has been confirmed by molecular docking of β-cyclodextrin to Gt-Mamy that indicated the requirement of homodimer formation by the interaction of a few N-terminal residues of chain A with the catalytic residues of (α/β)8 barrel of chain B and vice-versa for stable cyclodextrin binding. Site directed mutagenesis provided evidence for the role of N-terminal D109 at the dimeric interface in substrate affinity modulation and thermostabilization. The dimeric Gt-Mamy transglycosylates hydrolytic products of G4/G5 and acarbose, while the truncated form does not because of the lack of extra sugar-binding space formed due to dimerization.Conclusion/SignificanceN-terminal domain controls enthalpy-driven thermostabilization, substrate-binding affinity and transglycosylation activity of Gt-Mamy by homodimer formation.

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.