Monomer Choice Influences N-Acryloyl Amino Acid Grafter Conversion via Protease Catalysis.

End-capped peptides modified with reactive functional groups on the N-terminus provide a route to prepare peptide-polymer conjugates for a broad range of applications. Unfortunately, current chemical methods to construct modified peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks green preparative characteristics and is costly, thus limiting its applicability to specialty applications such as regenerative medicine. This work evaluates N-terminally modified N-acryloyl-glutamic acid diethyl ester, N-acryloyl-leucine ethyl ester, and N-acryloyl-alanine ethyl ester as grafters and papain as the protease for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease-catalyzed peptide synthesis (PCPS) and the corresponding formation of N-acryloyl-functionalized oligopeptides in a one-pot aqueous reaction. It was hypothesized that by building N-acryloyl grafters from AA-OEt monomers that are known to be good substrates for papain in PCPS, the corresponding grafters would yield high grafter conversions, high ratio of grafter-oligopeptide to free NH2-oligopeptide, and high overall yield. However, this work demonstrates based on the grafter/monomers studied herein that the dominant factor in N-acryloyl-AA-OEt grafter conversion is the co-monomer used in co-oligomerizations. Computational modeling using Rosetta qualitatively recapitulates the results and provides insight into the structural and energetic bases underlying substrate selectivity. The findings herein expand our knowledge of factors that determine the efficiency of preparing N-acryloyl-terminated oligopeptides by PCPS that could provide practical routes to peptide macromers for conjugation to polymers and surfaces for a broad range of applications.

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models.

Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

Lipase-Catalyzed Synthesis and Characterization of Poly(glycerol sebacate).

This study demonstrated that immobilized Candida antarctica lipase B (N435) catalysis in bulk leads to higher molecular weight poly(glycerol sebacate), PGS, than self-catalyzed condensation polymerization. Since the glass-transition temperature, fragility, modulus, and strength for rubbery networks are inversely dependent on the concentration of chain ends, higher molecular weight PGS prepolymers will enable the preparation of cross-linked PGS matrices with unique mechanical properties. The evolution of molecular species during the prepolymerization step conducted at 120 °C for 24 h, prior to enzyme addition, revealed regular decreases in sebacic acid and glycerol-sebacate dimer with corresponding increases in oligomers with chain lengths from 3 to 7 units such that a homogeneous liquid substrate has resulted. At 67 h, for N435-catalyzed PGS synthesis, the carboxylic acid conversion reached 82% without formation of a gel fraction, and number-average molecular weight (Mn) and weight-average molecular weight (Mw) values reached 6000 and 59 400 g/mol, respectively. In contrast, self-catalyzed PGS condensation polymerizations required termination at 55 h to avoid gelation, reached 72% conversion, and Mn and Mw values of 2600 and 13 800 g/mol, respectively. We also report the extent that solvent fractionation can enrich PGS in higher molecular weight chains. The use of methanol as a nonsolvent increased Mn and Mw by 131.7 and 18.3%, respectively, and narrower dispersity (D) decreased by 47.7% relative to the nonfractionated product.

Lipase-Catalyzed Poly(glycerol-1,8-octanediol-sebacate): Biomaterial Engineering by Combining Compositional and Crosslinking Variables.

This study examined poly(glycerol-1,8-octanediol-sebacate) (PGOS) copolymers with low-level substitution of O (1,8-octanediol) for G (glycerol) units (G/O ratios 0.5:0.5, 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09) prepared in bulk by immobilized Candida antarctica Lipase B (N435) catalysis. The central question explored was the extent that exchanging less than half of poly(glycerol sebacate) (PGS) glycerol units with 1,8-octanediol can be used as a strategy to fine-tune biomaterial properties. Synthesized copolymers having G/O ratios of 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09 have similar molecular weights, where Mw varied from 52,800 to 63,800 g/mol, Mn varied from 5100 to 6450 g/mol, and DM (molecular mass dispersity, Mw/Mn) values were also similar (8.4-11.4). All of the copolymers were branched, and dendritic glycerol units reached 11% for PGOS-0.91:0.09:1.0. Analysis of DSC second heating scans revealed that copolymers with higher 1,8-octanediol contents have relatively higher Tm and ΔHf values. Over the copolymer compositional range studied herein, Tm and ΔHf values varied from 5.3 to 21.1 °C and 8.0 to 23.1 J/g, respectively. Stress-strain curves of PGOS copolymers cured at 140 °C for 48 h exhibited either a unimodal, bimodal, or trimodal response to tensile loading. Varying G/O from 10:1 to 2:1 resulted in significant increases in the peak stress (0.26-4.01 MPa), preyield modulus (0.65-62.59 MPa), failure to strain (64-110%), and failure toughness (0.1-0.56 MPa). This demonstrates that altering the G/O ratio over a narrow compositional range provides biomaterials with widely different yet tunable mechanical properties. Further investigation of PGOS-0.75:0.25:1.0 films revealed that varying the cure conditions from 120 to 160 °C for periods of 24-72 h provides access to biomaterials with a failure strain range from 15 to 224% and Young's modulus from 1.17 to 10.85 MPa. Hence, using the dual variables of compositional variation and changes in cure conditions provides an exciting platform for PGS analogues to optimize material-tissue interactions. Increased contents of 1,8-octanediol slowed in vitro degradation. Slowed degradation of PGOS relative to PGS will be valuable for use in slower healing wounds.

Sophorolipids: Anti-cancer activities and mechanisms.

Sophorolipids (SLs) are biosurfactants synthesized as secondary metabolites by non-pathogenic yeasts and other microorganisms. They are members of glycolipid microbial surfactant family that consists of a sophorose polar head group and, most often, an ω-1 hydroxylated fatty acid glycosidically linked to the sophorose moiety. Since the fermentative production of SLs is high (>200 g/L), SLs have the potential to provide low-cost therapeutics. Natural and modified SLs possess anti-cancer activity against a wide range of cancer cell lines such as those derived from breast, cervical, colon, liver, brain, and the pancreas. Corresponding data on their cytotoxicity against noncancerous cell lines including human embryo kidney, umbilical vein, and mouse fibroblasts is also discussed. These results are compiled to elucidate trends in SL-structures that lead to higher efficacy against cancer cell lines and lower cytotoxicity for normal cell lines. While extrapolation of these results provides some insights into the design of SLs with optimal therapeutic indices, we also provide a critical assessment of gaps and inconsistencies in the literature as well as the lack of data connecting structure-to-anticancer and cytotoxicity on normal cells. Furthermore, SL-mechanism of action against cancer cell lines, that includes proliferation inhibition, induction of apoptosis, membrane disruption and mitochondria mediated pathways are discussed. Perspectives on future research to develop SL anticancer therapeutics is discussed.

Rapid Synthesis of Silk-Like Polymers Facilitated by Microwave Irradiation and Click Chemistry.

Silk is a natural fiber that surpasses most man-made polymers in its combination of strength and toughness. Silk fibroin, the primary protein component of silk, can be synthetically mimicked by a linear copolymer with alternating rigid and soft segments. Strategies for chemical synthesis of such silk-like polymers have persistently resulted in poor sequence control, long reaction times, and low molecular weights. Here, we present a two-stage approach for rapidly synthesizing silk-like polymers with precisely defined rigid blocks. This approach utilizes solid-phase peptide synthesis to create uniform oligoalanine "prepolymers", followed by microwave-assisted step-growth polymerization with bifunctional poly(ethylene glycol). Multiple coupling chemistries and reaction conditions were explored, with microwave-assisted click chemistry yielding polymers with Mw ∼ 14 kg/mol in less than 20 min. These polymers formed antiparallel ß-sheets and nanofibers, which is consistent with the structure of natural silk fibroin. Thus, our strategy demonstrates a promising modular approach for synthesizing silk-like polymers.

Structure-Activity Relationship Assessment of Sophorolipid Ester Derivatives against Model Bacteria Strains.

Sophorolipids (SLs) are glycolipids that consist of a hydrophilic sophorose head group covalently linked to a hydrophobic fatty acid tail. They are produced by fermentation of non-pathogenic yeasts such as Candida Bombicola. The fermentation products predominantly consist of the diacetylated lactonic form that coexists with the open-chain acidic form. A systematic series of modified SLs were prepared by ring opening of natural lactonic SL with n-alkanols of varying chain length under alkaline conditions and lipase-selective acetylation of sophorose primary hydroxyl groups. The antimicrobial activity of modified SLs against Gram-positive human pathogens was a function of the n-alkanol length, as well as the degree of sophorose acetylation at the primary hydroxyl sites. Modified SLs were identified with promising antimicrobial activities against Gram-positive human pathogens with moderate selectivity (therapeutic index, TI = EC50/MICB. cereus = 6-33). SL-butyl ester exhibited the best antimicrobial activity (MIC = 12 µM) and selectivity (TI = 33) among all SLs tested. Kinetic studies revealed that SL-ester derivatives kill B. cereus in a time-dependent manner resulting in greater than a 3-log reduction in cell number within 1 h at 2×MIC. In contrast, lactonic SL required 3 h to achieve the same efficiency.

Emergent Approaches to Efficient and Sustainable Polyhydroxyalkanoate Production.

Petroleum-derived plastics dominate currently used plastic materials. These plastics are derived from finite fossil carbon sources and were not designed for recycling or biodegradation. With the ever-increasing quantities of plastic wastes entering landfills and polluting our environment, there is an urgent need for fundamental change. One component to that change is developing cost-effective plastics derived from readily renewable resources that offer chemical or biological recycling and can be designed to have properties that not only allow the replacement of current plastics but also offer new application opportunities. Polyhydroxyalkanoates (PHAs) remain a promising candidate for commodity bioplastic production, despite the many decades of efforts by academicians and industrial scientists that have not yet achieved that goal. This article focuses on defining obstacles and solutions to overcome cost-performance metrics that are not sufficiently competitive with current commodity thermoplastics. To that end, this review describes various process innovations that build on fed-batch and semi-continuous modes of operation as well as methods that lead to high cell density cultivations. Also, we discuss work to move from costly to lower cost substrates such as lignocellulose-derived hydrolysates, metabolic engineering of organisms that provide higher substrate conversion rates, the potential of halophiles to provide low-cost platforms in non-sterile environments for PHA formation, and work that uses mixed culture strategies to overcome obstacles of using waste substrates. We also describe historical problems and potential solutions to downstream processing for PHA isolation that, along with feedstock costs, have been an Achilles heel towards the realization of cost-efficient processes. Finally, future directions for efficient PHA production and relevant structural variations are discussed.

Effect of Atomized Delivery of Nutrients on the Growth Characteristics and Microstructure Morphology of Bacterial Cellulose.

This work demonstrates a general strategy for introducing remarkable changes in matrix organization and, consequently, functional properties of bacterial cellulose (BC). BC-producing cells were induced, using a well-defined atomized droplet nutrient delivery (ADND) system, to form pellicles with a regular layered morphology that persists throughout the mat depth. In contrast, the morphology of mats formed by conventional static medium nutrient delivery (SMND) is irregular with no distinguishable pattern. ADND also resulted in larger meso-scale average pore sizes but did not alter the fibril diameter (∼70 nm) and crystallinity index (92-95%). The specific modulus and specific tensile strength of ADND mats are higher than those of SMND mats. This is due to the regularity of dense layers that are present in ADND mats that are able to sustain tensile loads, when applied parallel to these layers. The density of BC films prepared by ADND is 1.63-fold lower than that of the SMND BC film. Consequently, the water contents (g/g) of ADND- and SMND-prepared BC mats are 263 ± 8.85 and 99.6 ± 2.04, respectively. A model that rationalizes differences in mat morphology resulting from these nutrient delivery methods based on nutrient and oxygen concentration gradients is proposed. This work raises questions as to the extent that ADND can be used to fine-tune the matrix morphology and how the resulting lower density mats will alter the diffusion of actives from the films to wound sites and increase the ability of cells to infiltrate the matrix during tissue engineering.

Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)x.

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ ß-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Enzymatic Polymerization of Poly(glycerol-1,8-octanediol-sebacate): Versatile Poly(glycerol sebacate) Analogues that Form Monocomponent Biodegradable Fiber Scaffolds.

A family of poly(glycerol sebacate) (PGS) analogues were synthesized by Candida antarctica lipase B (CALB) catalysis to tailor biomaterial properties. Different fractions of glycerol (G) units in PGS were replaced by 1,8-octanediol (O) units. Poly(glycerol-1,8-octanediol-sebacate), PGOS, synthesized by CALB catalysis with a 1:3 molar ratio of G to O units has Mn and Mw values of 9500 and 92,000, respectively. PGS undergoes fiber fusion during electrospinning, and cross-linked PGS rapidly resorbs when implanted. By decreasing the molar ratio of glycerol-to-octanediol from 1:1 to 1:4, the peak melting temperature (Tm) increased from 27 to 47 °C. PGOS with 1:3 G to O units was electrospun into nanofibers without the need for a second component. The copolymer is semicrystalline and, when cross-linked, undergoes slow in vitro mass loss (3.5 ± 1.0% in 31 days) at pH 7.4 and 37 °C. Furthermore, PGOS cross-linked films have an elastic modulus of 106.1 ± 18.6 MPa, which is more than 100 times that of cross-linked PGS. New PGOS polymers showed tunable molecular weights, better thermal properties, and excellent electrospinnability. This work expanded PGS analogues' function, making these suitable biodegradable polymers for various biomedical applications.

Lipase-Catalyzed Reactive Extrusion: Copolymerization of ε-Caprolactone and ω-Pentadecalactone.

This study assesses the use of immobilized lipase catalyst N435 during reactive extrusion (REX) versus magnetically stirred bulk and solution reaction conditions for the copolymerization of ε-caprolactone with ω-pentadecalactone (CL/PDL 1:1 molar). N435-catalyzed REX for reaction times from 1 to 3 h results in total %-monomer conversion, Mn , and D values increase from 92.7% to 98.8%, 36.1 to 51.3 kDa, and 1.85 to 1.96, respectively. Diad fraction analysis by quantitative 13 C NMR reveals that, after just 1 h, rapid N435-catalyzed transesterification reactions occur that give random copolyesters. In contrast, for bulk polymerization with magnetic stirring in round bottom flasks, reaction times from 1 to 3 h result in the following: Mn  increases from 12.4 to 25.6 kDa, D decreases from 2.98 to 1.87, and the randomness index increases from 0.74 and 0.86 as PDL*-PDL diads are dominant. These results highlight that REX avoids problems associated with internal batch mixing that are encountered in bulk polymerizations. In sharp contrast to a previous study of 1:1 molar PDL/δ-valerolactone (VL) copolymerizations by N435-catalyzed REX, VL %-conversion increases to just 40.1% in 1 h whereas CL reaches 94.7%.

Engineering Corynebacterium glutamicum for the de novo biosynthesis of tailored poly-γ-glutamic acid.

γ-Polyglutamic acid (γ-PGA) is a biodegradable polymer naturally produced by Bacillus spp. that has wide applications. Fermentation of γ-PGA using Bacillus species often requires the supplementation of L-glutamic acid, which greatly increases the overall cost. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing γ-PGA from glucose. The genes encoding γ-PGA synthase complex from B. subtilis (pgsB, C, and A) or B. licheniformis (capB, C, and A) were expressed under inducible promoter Ptac in a L-glutamic acid producer C. glutamicum ATCC 13032, which led to low levels of γ-PGA production. Subsequently, C. glutamicum F343 with a strong L-glutamic acid production capability was tested. C. glutamicum F343 carrying capBCA produced γ-PGA up to 11.4 g/L, showing a higher titer compared with C. glutamicum F343 expressing pgsBCA. By introducing B. subtilis glutamate racemase gene racE under Ptac promoter mutants with different expression strength, the percentage of L-glutamic acid units in γ-PGA could be adjusted from 97.1% to 36.9%, and stayed constant during the fermentation process, while the γ-PGA titer reached 21.3 g/L under optimal initial glucose concentrations. The molecular weight (Mw) of γ-PGA in the engineered strains ranged from 2000 to 4000 kDa. This work provides a foundation for the development of sustainable and cost-effective de novo production of γ-PGA from glucose with customized ratios of L-glutamic acid in C. glutamicum.

Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis.

Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.

Grown Ultrathin Bacterial Cellulose Mats for Optical Applications.

A facile and effective method is described for the biosynthesis of ultrathin bacterial cellulose (BC) mats, which are green, inexpensive, lightweight, and flexible. Physical properties studied include thickness, morphology, reflectance, transmittance, and crystallinity index. BC mat thickness was varied by controlling the depth of the culture broth so that films with predictable thickness, between 113 and 1114 nm, were produced. These BC films have similar fiber morphology to corresponding mm thick BC films prepared under static culture conditions. To increase BC film hydrophobicity, surface trihexylsilylated BC (THSBC) mats with DSavg 0.015 were prepared. Both native and THSBC mats were investigated as antireflection coatings for silicon substrates. The 328 ± 42 nm thick BC mat demonstrated broadband, interference type antireflection over a spectral range of 500-1800 nm. Different reflection properties obtained as a function of BC film orientation reveals that engineered density gradients can be used to manipulate BC optical properties. Thus, optical quality and environmental friendly ultrathin BC films are promising biomaterials for next-generation optoelectronic devices.

Biosurfactant-functionalized porphyrin chromophore that forms J-aggregates.

Structurally complex biosynthesized building blocks whose structures can be systematically varied are of great interest for the synthesis of manipulable self-organizing supramolecular systems. Sophorolipids (SLs) are an important class of glycolipid biosurfactants that consists of a sophorose (glucose disaccharide) polar head group that allows structural diversification by full or selective acetylation at the 6'- and 6''-positions. Porphyrins are a group of naturally-occurring heterocyclic macromolecular organic compounds that have efficient charge transfer properties. Herein we describe the synthesis of SL-porphyrin conjugates where the number of sophorolipid arms, availability of hydrogen bonding sophorose hydroxyl groups and rigidity of the lipid chain were systematically varied. SLs differing in 'sophorose acetylation' and 'lipid unsaturation' were conjugated to zinc-porphyrin dyes by copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) 'click' chemistry. Mono-, di-, and tetra-conjugation of SL-arms to the zinc-porphyrin core provided variation in SL-arm steric effects. UV-vis spectra in methanol/water reveal features indicative of supramolecular J-type aggregates. The synthesized compounds were designed to provide a library of unique bio-based molecules with built-in variation in non-covalent interactions, hydrogen-bonding, π-π stacking, metal-ligand coordination, dipole-dipole, van der Waals, and hydrophobic interactions for future interrogation of supramolecular self-assembly into functional materials for electro-optical applications.

Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.

Fundamental Characterization of the Micellar Self-Assembly of Sophorolipid Esters.

Surfactants are ubiquitous constituents of commercial and biological systems that function based on complex structure-dependent interactions. Sophorolipid (SL) n-alkyl esters (SL-esters) comprise a group of modified naturally derived glycolipids from Candida bombicola. Herein, micellar self-assembly behavior as a function of SL-ester chain length was studied. Surface tensions as low as 31.2 mN/m and critical micelle concentrations (CMCs) as low as 1.1 µM were attained for diacetylated SL-decyl ester (dASL-DE) and SL-octyl ester, respectively. For deacetylated SL-esters, CMC values reach a lower limit at SL-ester chains above n-butyl (SL-BE, 1-3 µM). This behavior of SL-esters with increasing hydrophobic tail length is unlike other known surfactants. Diffusion-ordered spectroscopy (DOSY) and T1 relaxation NMR experiments indicate this behavior is due to a change in intramolecular interactions, which impedes the self-assembly of SL-esters with chain lengths above SL-BE. This hypothesis is supported by micellar thermodynamics where a disruption in trends occurs at n-alkyl ester chain lengths above those of SL-BE and SL-hexyl ester (SL-HE). Diacetylated (dA) SL-esters exhibit an even more unusual trend in that CMC increases from 1.75 to 815 µM for SL-ester chain lengths of dASL-BE and dASL-DE, respectively. Foaming studies, performed to reveal the macroscopic implications of SL-ester micellar behavior, show that the observed instability in foams formed using SL-esters are due to coalescence, which highlights the importance of understanding intermicellar interactions. This work reveals that SL-esters are an important new family of green high-performing surfactants with unique structure-property relationships that can be tuned to optimize micellar characteristics.

Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris.

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.

Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris.

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.