Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.     

Salimyxins and Enhygrolides: Antibiotic,Sponge‐Related Metabolites from the Obligate Marine Myxobacterium Enhygromyxa salina

Unlike their terrestrial counterparts, marine myxobacteria are hardly investigated for their secondary metabolites. This study describes three new compounds ( 1 – 3 ), named salimyxins and enhygrolides, obtained from the obligate marine myxobacterium Enhygromyxa salina. These are the first natural products obtained from Enhygromyxa species. Their structures were elucidated by spectroscopic analysis, including NMR and CD spectroscopy. Enhygrolides are closely related to the nostoclides, which were initially isolated from a cyanobacterium of the genus Nostoc. The salimyxins, representing structurally most unusual degraded sterols, are close to identical to demethylincisterol from the sponge Homaxinella sp. Salimyxin B and enhygrolide A inhibit the growth of the Gram‐positive bacterium Arthrobacter cristallopoietes (MIC salimyxin B, 8 μg mL^?1; enhygrolide A, 4 μg mL^?1).     

Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase‐1 (DDAH‐1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N⁵‐(1‐imino‐2‐chloroethyl)‐L ‐ornithine (Cl‐NIO) is shown to be a potent time‐ and concentration‐dependent inhibitor of purified human DDAH‐1 (K_I=1.3±0.6 μM ; k_inact=0.34±0.07 min^?1), with >500‐fold selectivity against two arginine‐handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC₅₀ value (6.6±0.2 μM ) for Cl‐NIO inhibition of DDAH‐1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH‐1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl‐NIO shows a subsequent decrease in cellular nitric oxide production. Cl‐NIO is a promising tool for the study of methylarginine‐mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.     

E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain‐like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule‐associated serine proteases. Its exopeptidase activity is structurally explained by the so‐called exclusion domain, which blocks the active‐site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)‐trans‐epoxysuccinyl‐L ‐leucylamido‐3‐methylbutane (E‐64c) (k₂/K_i=140±5 M ^?1 s^?1) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′–S2′ area with its leucine‐isoamylamide moiety. Furthermore, structure–activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n‐butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56 000±1700 M ^?1 s^?1).     

Experimental design of the kinetic resolution of a key precursor of high‐value bioactive myo‐inositols by an immobilized lipase

BACKGROUND: The response surface methodology was successfully applied to the optimization of the reaction variables for the kinetic resolution of a precursor of high‐value myo‐inositols, ( ± )‐1,2‐O‐isopropylidene‐3,6‐di‐O‐benzyl‐myo‐inositol (( ± )‐1), by Novozym 435. The resolutions were run separately, with two acylating agents, ethyl acetate and vinyl acetate, in a solvent‐free system. The variables analyzed were reaction temperature, substrate concentration, water concentration and enzyme activity. A statistical model was employed for the evaluation of the influence of the variables on conversion and enantiomeric excess (ee). RESULTS: The optimal conditions for this resolution using vinyl acetate as acylating agent were 45 °C, 5 mg mL^?1 of substrate, 71 U of enzyme activity and 0%w/w of water concentration. The high conversion (49.2 %) and ee (>99%) reached in the chemoenzymatic synthesis of acylated product, L‐(?)‐5‐O‐Acetyl‐3,6‐di‐O‐benzyl‐1,2‐O‐isopropylidene‐myo‐inositol, secure the efficient synthesis of the D enantiomorph present in the original racemic mixture (( ± )‐1) as well. CONCLUSIONS: The use of the experimental design strategy was productive, leading to a 14‐fold increase in the productivity of the reaction compared with the non‐optimized conditions. Both derivative L‐(?)‐2 and remaining substrate D‐(+)‐1 were obtained at high ee. © 2012 Society of Chemical Industry     

DNA adsorption on a poly‐L‐lysine‐immobilized poly(2‐hydroxyethyl methacrylate) membrane

The DNA adsorption properties of poly‐L ‐lysine‐immobilized poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane were investigated. The pHEMA membrane was prepared by UV‐initiated photopolymerization and activated with epichlorohydrin. Poly‐L ‐lysine was then immobilized on the activated pHEMA membrane by covalent bonding, via a direct chemical reaction between the amino group of poly‐L ‐lysine and the epoxy group of pHEMA. The poly‐L ‐lysine content of the membrane was determined as 1537 mg m^?2. The poly‐L ‐lysine‐immobilized membrane was utilized as an adsorbent in DNA adsorption experiments. The maximum adsorption of DNA on the poly‐L ‐lysine‐immobilized pHEMA membrane was observed at 4 °C from phosphate‐buffered salt solution (pH 7.4, 0.1 M; NaCl 0.5 M) containing different amounts of DNA. The non‐specific adsorption of DNA on the plain pHEMA membrane was low (about 263 mg m^?2). Higher DNA adsorption values (up to 5849 mg m^?2) were obtained in which the poly‐L ‐lysine‐immobilized pHEMA membrane was used. Copyright © 2003 Society of Chemical Industry     

Novel Antitumor L‐Arabinose Derivative of Indolocarbazole with High Affinity to DNA

Novel indolocarbazole derivative 12‐(α‐L ‐arabinopyranosyl)indolo[2,3‐α]pyrrolo[3,4‐c]carbazole‐5,7‐dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)₂₀, d(GC)₂₀ and calf thymus DNA (binding constants (1.6×10⁶) M ^?1≤K_a≤(3.3×10⁶) M ^?1). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C≤1 μM ) were identical to the concentrations that triggered p53‐dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I‐mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.     

Synthesis,physical properties,and crystallization of optically active poly(L‐phenyllactic acid) and poly(L‐phenyllactic acid‐co‐L‐lactic acid)

Optically active poly(L ‐phenyllactic acid) (Ph‐PLLA), poly(L ‐lactic acid) (PLLA), and poly(L ‐phenyllactic acid‐co‐L ‐lactic acid) with weight‐average molecular weight exceeding 6 × 10³ g mol^?1 were successfully synthesized by acid catalyzed direct polycondensation of L ‐phenyllactic acid and/or L ‐lactic acid in the presence of 2.5–10 wt % of p‐toluenesulfonic acid. Their physical properties and crystallization behavior were investigated by differential scanning calorimetry, thermogravimetry, and polarimetry. The absolute value of specific optical rotation ([α]) for Ph‐PLLA (?38 deg dm^?1 g^?1 cm³) was much lower than that of [α] for PLLA (?150 deg dm^?1 g^?1 cm³), suggesting that the helical nature was reduced by incorporation of bulky phenyl group. PLLA was crystallizable during solvent evaporation, heating from room temperature, and cooling from the melt. Incorporation of a very low content of bulky phenyllactyl units even at 4 mol % suppressed the crystallization of L ‐lactyl unit sequences during heating and cooling, though the copolymers were crystallizable for L ‐phenylactyl units up to 6 mol % during solvent evaporation. The activation energy of thermal degradation (ΔE_td) for Ph‐PLLA (200 kJ mol^?1) was higher than that for PLLA (158 kJ mol^?1). The ΔE_td for the copolymers increased with an increase in L ‐phenyllactyl unit content. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

In silico Optimization of a Fragment‐Based Hit Yields Biologically Active,High‐Efficiency Inhibitors for Glutamate Racemase

A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase (GR), was optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid molecular dynamics–docking and scoring scheme, FERM‐SMD, was used to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high‐ and low‐affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency was increased approximately fourfold against GR from the model organism, B. subtilis. Lead derivatives show two‐ to fourfold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis over E. coli and S. aureus depends on the substituent added to the parent scaffold. Finally, insight was gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The outcome of this study is a novel small‐molecule inhibitor of GR with the following characteristics: K_i=2.5 μM , LE=0.45 kcal mol^?1 atom^?1, LiPE=6.0, MIC₅₀=260 μg mL^?1 against B. subtilis, EC_{50, lysis}=520 μg mL^?1 against B. subtilis.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Targeting Cystalysin,a Virulence Factor of Treponema denticola‐Supported Periodontitis

Cystalysin from Treponema denticola is a pyridoxal 5′‐phosphate dependent lyase that catalyzes the formation of pyruvate, ammonia, and sulfide from cysteine. It is a virulence factor in adult periodontitis because its reaction contributes to hemolysis, which sustains the pathogen. Therefore, it was proposed as a potential antimicrobial target. To identify specific inhibitors by structure‐based in silico methods, we first validated the crystal structure of cystalysin as a reliable starting point for the design of ligands. By using single‐crystal absorption microspectrophotometry, we found that the enzyme in the crystalline state, with respect to that in solution, exhibits: 1) the same absorption spectra for the catalytic intermediates, 2) a close pK_a value for the residue controlling the keto enamine ionization, and 3) similar reactivity with glycine, L ‐serine, L ‐methionine, and the nonspecific irreversible inhibitor aminoethoxyvinylglycine. Next, we screened in silico a library of 9357 compounds with the Fingerprints for Ligands and Proteins (FLAP) software, by using the three‐dimensional structure of cystalysin as a template. From the library, 17 compounds were selected and experimentally evaluated by enzyme assays and spectroscopic methods. Two compounds were found to competitively inhibit recombinant T. denticola cystalysin, with inhibition constant (K_i) values of 25 and 37 μM . One of them exhibited a minimum inhibitory concentration (MIC) value of 64 μg mL^?1 on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.     

Novel 1,4‐Substituted‐1,2,3‐Triazoles as Antitubercular Agents

Tuberculosis (TB) remains a pressing unmet medical need, particularly with the emergence of multidrug‐resistant and extensively drug‐resistant tuberculosis. Here, a series of 1,4‐substituted‐1,2,3‐triazoles have been synthesized and evaluated as potential antitubercular agents. These compounds were assembled via click chemistry in high crude purity and in moderate to high yield. Of the compounds tested, 12 compounds showed promising antitubercular activity with six possessing minimum inhibitory concentration (MIC) values <10 μg mL^?1, and total selectivity for Mycobacterium tuberculosis (Mtb) growth inhibition. A second set of 21 compounds bearing variations on ring C were synthesized and evaluated. This second library gave an additional six compounds displaying MIC values ≤10 μg mL^?1 and total selectivity for Mtb growth inhibition. These compounds serve as an excellent starting point for further development of antitubercular therapies.     

Ammonium fumarate production by free or immobilised Rhizopus arrhizus in bench‐ and laboratory‐scale bioreactors

Ammonium fumarate production from glucose‐based media by Rhizopus arrhizus NRRL 1526 with mycelial growth controlled by phosphorus limitation exhibited mixed‐growth‐associated product formation kinetics, with growth‐associated production related to secondary mycelial growth only. The contribution of the primary mycelial growth phase was minimised by resorting to prolonged batch production using free mycelia under intermittent glucose feeding or repeated batch production using immobilised mycelia. The metabolic activity of free or immobilised mycelia was limited by fumarate accumulation or by oxygen diffusion phenomena, respectively. For batch cultures in a 15 dm³ stirred bioreactor the peripheral impeller speed (v_I) was increased from 1.88 to 3.3 m s^?1, and the fumarate yield coefficient on glucose increased from 0.25 ± 0.01 to 0.42 ± 0.02 g g^?1, while the malate yield coefficient on fumarate (Y_M/F) reduced from 0.46 ± 0.01 to 0.14 ± 0.01 g g^?1. With a net increase in the fumarate‐to‐malate ratio from 2 to 6.5, a v_I value of 3.3 m s^?1 gave the best fermentation performance and provided a basis for further scale‐up studies. © 2002 Society of Chemical Industry     

Inhibition of Bacterial Dihydrofolate Reductase by 6‐Alkyl‐2,4‐diaminopyrimidines

(±)‐6‐Alkyl‐2,4‐diaminopyrimidine‐based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies revealed attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125–8 μg mL^?1 range for both organisms. This effect was rationalized from three‐ dimensional X‐ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6, the main interactions are with protein residues Leu 20 and Leu 28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these water molecules such that it becomes continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction, highlighting a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.     

Design of a probe based on poly(glycidyl methacrylate‐co‐vinylferrocene)‐coated Pt electrode for electrochemical detection of PTEN gene in PCR amplified samples from prostate tissues

In this study, a new probe based on immobilization of amino linked oligonucleotide (NH₂‐linked DNA) on poly(glycidyl methacrylate‐co‐vinylferrocene)‐coated Pt electrode was fabricated for the electrochemical detection of PTEN gene from human prostate tissues. The experimental parameters such as DNA immobilization time, DNA concentration, and target concentration were optimized. The selectivity of the NH₂‐linked DNA probe was assessed with mismatch (MM) and noncomplementary (NC) sequences. The applicability of the NH₂‐linked DNA probe to the PCR amplified samples correspond to PTEN gene from prostate tissues was evaluated. The immobilization of DNA on the copolymer was confirmed by FTIR, AFM, CV and DPV analysis. The PCR products were also identified by using agarose gel electrophoresis. The prepared probe indicated a linear range (10–100 μg mL^?1) with a detection limit (4.7 μg mL^?1) and a good selectivity of the NH₂‐linked DNA probe toward target DNA sequence. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40638.     

Synthesis,characterization, and application of a new chelating resin functionalized with dithiooxamide

Chloromethylated polystyrene‐divinylbenzene has been functionalized with dithiooxamide. The resulting chelating resin (DTOA) has been characterized by elemental analyses, infrared spectroscopy, thermogravimetric analysis, and metal ion sorption capacities. It has been used for the preconcentration and separation of Cu(II), Zn(II), Cd(II), and Pb(II) prior to their determination by FAAS. Parameters such as the amount of the resin, effect of pH, equilibration rate, sorption and desorption of metal ions, and effect of diverse ions have been studied. The maximum sorption capacities found are 0.97, 0.12, 0.08, and 0.12 mmol g^?1 for Cu(II), Zn(II), Cd(II), and Pb(II) at pH 6.0, 5.5, 1.0, and 5.5, respectively. The preconcentration factors are 100, 100, 50, and 50 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. Recoveries of the metal ions were 96 ± 5, 97 ± 6, 96 ± 5, and 96 ± 5 at 95% confidence level, whereas the limits of detection are 2.0, 1.3, 2.5, and 25.0 μg L^?1 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The calibration curves were linear up to 12 μg mL^?1 (R² = 1.000), 2 μg mL^?1 (R² = 0.998), 2 μg ml^?1 (R² = 1.000), and 5 μg mL^?1 (R² = 0.979) for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The reliability of the method has been tested by analyzing certified samples. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2281–2285, 2007     

Efficient Whole‐Cell Biocatalytic Synthesis of N‐Acetyl‐D‐neuraminic Acid

N‐Acetyl‐D ‐neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N‐acetyl‐D ‐glucosamine (GlcNAc) and N‐acetyl‐D ‐mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32±0.56 g/liter Neu5Ac were obtained from 65.61±2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4±0.4 % purity, 80.87±0.79 % recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes.     

High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his‐tagged rhamnulose‐1‐phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm^?3 and 288 AU dm^?3 h^?1 respectively) compared with Luria–Bertoni (LB) medium (2292 AU dm^? 3 and 255 AU dm^?3 h^?1). The minimum quantity of (isopropyl‐β‐D ‐thiogalactoside) IPTG for optimal induction was estimated in 18–20 µmol IPTG gDCW^?1. The highest volumetric production of RhuA (8333 AU dm^?3) was obtained when IPTG was added in the late log‐phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37 °C. An effective two‐step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications. Copyright © 2003 Society of Chemical Industry     

Non‐isothermal crystallization behavior of stereo diblock polylactides with relatively short poly(d‐lactide) segments from the melt

Stereo diblock polylactides (SDB‐PLAs) composed of relatively short poly(d ‐lactide) (PDLA) segments and relatively long poly(l ‐lactide) (PLLA) segments were synthesized to have a wide number‐average molecular weight (M_n) range of 2.5 × 10⁴–2.0 × 10⁵ g mol^?1 and d ‐lactyl unit content of 0.9–38.6%. The effects of incorporated short PDLA segments (M_n = 2.0 × 10³–7.7 × 10³ g mol^?1) on crystallization behavior of the SDB‐PLAs were first investigated during heating after complete melting and quenching or during slow cooling after complete melting. Stereocomplex (SC) crystallites can be formed at d ‐lactyl unit content as low as 4.3 and 5.8% for heating and slow cooling, respectively, and for M_n of PDLA segments as low as 2.0 × 10³ and 3.5 × 10³ g mol^?1, respectively. With decreasing M_n and increasing d ‐lactyl unit content, the cold crystallization temperature during heating decreased and the crystallization temperature during slow cooling increased. With increasing d ‐lactyl unit content, the melting enthalpy (ΔH_m) of SC crystallites during heating and the crystallinity (X_c) of SC crystallites after slow cooling increased, whereas ΔH_m of PLLA homo‐crystallites during heating and X_c of PLLA homo‐crystallites after slow cooling decreased. The total ΔH_m of SC crystallites and PLLA homo‐crystallites during heating and the total X_c after slow cooling became a minimum at d ‐lactyl unit content of 10–15% and gave a maximum at d ‐lactyl unit content of 0%. Despite the accelerated crystallization of some of SDB‐PLAs, the low values of total ΔH_m and X_c at d ‐lactyl unit content of 10–15% are attributable to the formation of two crystalline species of SC crystallites and PLLA homo‐crystallites.