A Comparison of Protease Active Sites and their Ability to Process Silicon-Based Substrates

Many enzymes have been identified that can participate in the hydrolysis of alkoxysilanes; each with a different degree of specificity. Our working hypothesis was that the nature of the active site of the enzyme (i.e., the compatibility of binding pockets with the substrate) could have a direct effect on the rate of catalysis. This communication reports our experiments on the relative rates of hydrolysis of a model alkoxysilane, phenyltrimethoxysilane (PTMS), by three proteases: trypsin, α-chymotrypsin, and pepsin. Trypsin which typically accepts amino acids bearing positively charged basic residues was not particularly proficient for the hydrolysis of PTMS. On the other hand, both α-chymotrypsin and pepsin, each of which contains a binding pocket, or two in the case of pepsin, suitable for accommodating aromatic residues, were more suitable for mediating hydrolysis. This report provides some preliminary data to support the hypothesis that the architecture of the enzyme active site is important in determining the proficiency with which an enzyme will process a given organosilicon substrate.     

Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4‐Oxalocrotonate Tautomerase More Effectively than Distant Mutations

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which catalyzes enol–keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon–carbon bond‐forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4‐OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000‐fold improvement in catalytic efficiency (k_cat/K_m) and a >10⁷‐fold change in reaction specificity, by exploring small libraries in which only “hotspots” are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4‐OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.     

Synthesis of polysiloxane-modified silica hybrid particles by a high temperature water technology

Polysiloxane-modified silica hybrid particles were synthesized by hydrolysis and subsequent polycondensation of phenyltrimethoxysilane in the presence of colloidal silica at above 220 °C. The process variables include pH of the colloid, [silica]/[phenyltrimethoxysilane] molar ratio, reaction temperature, and reaction time. The products were characterized by solvent extraction, TGA, GPC, TEM, and ²⁹Si NMR. The degree of surface modification (DOM) ranged between 5 and 35 wt.% by tuning the process variables when a neutral colloid was used. On the other hand, the DOM proved to be strongly dependent on temperature and becomes essentially zero when the synthesis was conducted with an acidic colloid at temperatures as high as 350 °C. TEM analysis suggested that each silica particle was surface modified resulting in minimal aggregation of the particles. The hybridization mechanism was studied by the DOM and the molecular weight of ungrafted polysiloxane.     

Immobilization and Activity of Pepsin in Silicone Elastomers

Pepsin [EC, 3.4.23.1] from Porcine stomach mucosa was immobilized in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). Two network precursor chain molar masses were used in this investigation: in pepsin–silicone (A), M _n ∼26,000 g mol⁻¹ and in pepsin–silicone (B) M _n ∼750 g mol⁻¹. Tetraethyl orthosilicate (TEOS) was used as the cross-linking agent and dibutyltin dilaurate was used as the catalyst. The activity and stability of free pepsin and pepsin immobilized in PDMS were studied with respect to pH, temperature, cross-link density, solvents and storage time using a hemoglobin assay. A notable finding is that free pepsin has zero activity in neutral buffer solution (pH 7) after incubation for 5 h, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. There was no marked improvement in the thermal stability of the PDMS immobilized pepsin when compared to free pepsin and all the three systems showed no activity at and above 70 °C. From the Lineweaver–Burk kinetic analyses, the apparent K _m (g L⁻¹ hemoglobin) for free pepsin was 4.5, for pepsin–silicone (A) was 5.1, and for pepsin–silicone (B) was 3.9, the V _max (U/mg of pepsin) for free pepsin was 14,000, for pepsin–silicone (A) was 11,710, and for pepsin–silicone (B) was 8,510, respectively after incubation in buffer solution at pH 2 and 37 °C. The activity of the free and the PDMS immobilized pepsin in six different organic solvents was also studied. The pepsin retained high activity in non-polar solvents such as n-hexane, isooctane and toluene, but the enzyme performed poorly in methanol, ethanol and tetrahydrofuran. The degree of swelling of the pepsin immobilized silicone elastomers in these solvents had no impact on the activity of the pepsin. When stored at room temperature for time periods up to 6 months, pepsin immobilized in silicone elastomers was observed to retain its full activity. The results reported herein demonstrate that cross-linked PDMS is a promising support material for the immobilization of hydrolytic enzymes such as pepsin.     

Chemoselective hydrolysis of methyl 2‐acetoxybenzoate using free and entrapped esterase in K‐carrageenan beads

Porcine liver esterase was entrapped in natural polysaccharides K‐carrageenan and retention of its activity was determined using p‐nitrophenyl acetate as the substrate. The optimum pH for esterase activity of entrapped enzyme showed a little shift towards acidic side. Immobilized enzyme showed improved thermal and storage stability. The entrapped esterase retained 50% of its activity after eight repetitive cycles. Michaelis constant K_m for the free and entrapped enzymes was almost same indicting no conformational change during immobilization. Maximum velocity V_max was observed to decrease on immobilization. The free and entrapped esterase was used for selective hydrolysis of methyl 2‐acetoxybenzoate to methyl 2‐hydroxybenzoate in batch process as well as in a fixed bed reactor. The hydrolysis was observed to be 99% within 2 h for free as well as immobilized enzyme in batch process. The rate of hydrolysis was found to depend on pH. The turn over number of selective hydrolysis in batch and fixed bed reactor was 3.08 × 10⁶ and 1.19 × 10⁷, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

聚二甲基硅氧烷—苯基倍半硅氧烷共聚物及其弹性体 Ⅱ共聚物流动性和硫化胶力学性能的影响因素

羟基封端的聚二甲基硅氧烷和苯基三甲氧基硅烷在有机溶剂中进行碱催化水解缩合反应，形成了聚二甲基硅氧烷—苯基倍半硅氧烷共聚物。在有机锡催化剂存在下，共聚物于室温可硫化成力学性能优良的有机硅弹性体。通过考察各种反应因素对共聚物流动性及硫化胶力学性能的影响，发现Ｈ２Ｏ与ＰｈＳｉ（ＯＣＨ３）３的摩尔比、１０７胶与ＰｈＳｉ（ＯＣＨ３）３的重量比、１０７胶分子量、甲苯用量对共聚物粘度和硫化胶力学性能有较大的影响，其中Ｈ２Ｏ与ＰｈＳｉ（ＯＣＨ３）３的摩尔比是决定共聚物及其硫化胶基本性能的关键因素。     

Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H⁺ and Na⁺ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a “direct coupling” mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na⁺ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K⁺-dependent H⁺-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PP_i analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PP_i hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg²⁺ (essential cofactor). The replacement of K⁺, the second metal cofactor, by Na⁺ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PP_i hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell’s direct coupling with conformational coupling to catalyze cation transport across the membrane.     

The effect of pretreatment on the enzymic hydrolysis of cellulosic industrial waste

The enzymic hydrolysis of cellulosic waste material from industrial origins using commercial Tricoderma viride cellulase gave yields approaching 80% conversion following pretreatments. The yield was a function of the purity of the substrate and the pretreatments imposed. These included sterilisation, heating, ball-milling and alkali treatment. Highest yields of glucose or enzyme hydrolysis were achieved with a 4% solka floc suspension following pretreatment in 5% alkali, when the enzyme mix was 0.22IU cm^?3 of filter paper activity, 0.07IU cm^?3C_x enzyme activity and 0.14IU cm^?3 of β-glucosidase activity. Kraft pulp also gave up to 75% yields by this method.     

Optimization of the enzymatic hydrolysis conditions of steam‐exploded wheat straw for maximum glucose and xylose recovery

BACKGROUND: The enzymatic hydrolysis of steam‐exploded wheat straw using commercial enzyme complexes has been studied. A cellulase enzyme complex (Accellerase 1500), along with specific xylanase complements (Accellerase‐XC and Accellerase‐XY) provided by Genencor, have been used to enhance glucose and xylose recovery. A systematic study with response surface methodology (RSM) was used to check the effect of the operating conditions: pH (4–5), temperature (50–60 °C) and enzyme/substrate ratio (0.1–0.5 mL g_cellulose^?1) on the enzymatic hydrolysis with Acellerase 1500 to maximize the sugar yield. Xylanases were used as complements to increase the release of xylose. RESULT: Statistical results from ANOVA analysis demonstrated that the enzymatic hydrolysis was clearly improved by temperature and enzyme/substrate ratio. The optimum conditions for higher glucose and xylose releases were obtained with the higher enzyme dosage ratio (0.5 mL g^?1_cellulose), 50 °C and pH 4. CONCLUSION: Model validation at optimum operating conditions showed good agreement between the experimental results and the predicted responses for a confidence level of 95%. The use of the xylanase complements, Accellerase‐XY (accessory xylanase enzyme complex) and Accellerase‐XC (accessory xylanase/cellulase enzyme complex), increases the conversion of hemicellulose. Accellerase‐XC supplementation was more effective, obtaining an increase in yields of glucose and xylose of 11.8% and 23.6%, respectively, using a dosage of 0.125 mL g^?1_cellulose. © 2012 Society of Chemical Industry     

Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates

In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora) hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8%) after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH) (56.00%) and ferrous ion-chelating (FIC) (59.00%) methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions.     

Hydrolysis and Condensation of Hydrophilic Alkoxysilanes Under Acidic Conditions

A hydrophilic silane was obtained from the reaction of ethylene carbonate and 3-aminopropyldiethoxymethylsilane. This silane undergoes rearrangement to yield an AB₂-type hyperbranched polymer under anhydrous conditions but hydrolyzes and condenses to produce linear siloxanes under acid hydrolysis. The hydrolysis and condensation reactions as a function of time, HCl concentration and water content were studied by ²⁹Si NMR. The compositions of the silanol containing hydrolysis intermediates and the siloxanes condensation products were identified under different conditions. The instantaneous composition was found to depend on the specific combination of the acid and the water. Under certain conditions the intermediate silane-diols were stable and did not condense even under mild acidic conditions.     

Control of hydroxyl group content in silica particle synthesized by the sol-precipitation process

This study investigated the control of hydroxyl groups, one of key factors determining the surface properties of silica particles synthesized by the sol-precipitation of tetraethyl orthosilicate (TEOS). Thus, a thermal gravity analysis (TGA) was used to facilitate quantitative measurements of the hydroxyl groups on the silica particles, while BET and FT-IR were used to analyze the specific surface area and functional silane groups on the silica particles, respectively. In the sol-precipitation process, silanes that include various hydroxyl groups are formed as intermediates based on the hydrolysis and condensation of TEOS. Thus, NH₃, as a basic catalyst initiating the nucleophilic substitution of TEOS, was found to accelerate the hydrolysis and increase the hydroxyl group content on the silica particles. Plus, the hydroxyl group content was also increased when increasing the concentrations of TEOS and water as the hydrolysis reactants. However, the hydroxyl group content was reduced when increasing the temperature, due to the promotion of condensation. Based on the weight loss of the particles according to the thermal analysis, the hydroxyl group content on the silica particles varied from 5.6–42.7 OH/nm² under the above reaction conditions.     

Synthesis of beta-SiC powder from alkoxide precursor and study of its sintering behaviour

Abstract

Abstract

In this study, β-SiC powder was prepared using a pyrolysed spherical precursor derived from the hydrolysis mixture of phenyltrimethoxysilane and tetraethyl orthosilicate. Before the pyrolysed experiment, an alkoxide precursor was characterised using ²⁹Si solid nuclear magnetic resonance, Fourier transform infrared spectroscopy and thermogravimetric analysis. The alkoxide precursor was heated at 1800°C for 4 h under an Ar atmosphere. To examine the pyrolysed residue after heat treatment, the sample was collected and analysed with X-ray diffraction. The X-ray diffraction results for the sample show diffraction peaks at ～35, 60 and 73°, which correspond to the β-SiC phase. According to the results of chemical analysis, the SiC content of the powder that was prepared at 1800°C was determined to be 99·4%. The sintering behaviour of the prepared β-SiC powder was examined using B₄C and C as sintering additives in the temperature range of 1900–2200°C.     

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

Poly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g^?1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (V_max) and Michaelis constant (K_m) of immobilized enzyme were found to be 0.74 μM s^?1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43343.     

Hydrolytic aging of polysiloxane networks modeling the glass fiber epoxy-amine interphase

It has previously been shown that polycondensates of trialkoxysilanes (AlkO)₃SiR could chemically simulate coupling agent layers located at the interphase of amine-crosslinked epoxy/glass fiber composites (1) and that a humid environment modifies the degree of condensation of the network. Although it is generally believed that water will inevitably hydrolyze the polysiloxane structure and destroy the interphase (2), the authors have demonstrated that the siloxane links of the network evolve toward an equilibrium state. This state depends on the chemical structure of the organic chain that can react with the matrix. For example, in the case of aminopropyltriethoxysilane, the siloxane equilibrium concentration is low enough to allow total hydrolysis of the polymer. Conversely, propylsilane network stability could be explained by a very high siloxane equilibrium concentration. In this article, one of the previously studied systems has been selected: GPS (glycidylpropylsilane), in which the coupling function is an epoxide group (glycidyl). In an epoxy/glass fiber composite, this group is expected to react with an amine group belonging to the epoxy network. Aniline has been used here to model the GPS-epoxy network bonding. This reaction modifies the chemical nature of the organic chain branched on the silicon and then potentially displaces the siloxane equilibrium. A gravimetric method, size exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FTIR), ²⁹Si nuclear magnetic resonance (NMR), and ¹³C NMR have been used. The results are that, when exposed to hot water, the GPS and the GPS-aniline networks evolve contradictorily. Although GPS tends to hydrolysis, GPS-aniline tends to condensation. This article analyzes the compatibility of the different behaviors with the simple kinetic model reported in a previous paper (1) and the importance of this phenomenon concerning the aging of the glass/matrix interphase of composite materials.     

Probing the Substrate Requirements of the In Vitro Geranylation Activity of Selenouridine Synthase (SelU)

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U₃₅ was mutated to A₃₅ (ASL-tRNA^Lys_(s2U)UU to ASL-tRNA^Ile_(s2U)AU). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASL^Lys_(s2UUU) ASL^Gln_(s2UUG)>ASL^Glu_(s2UUC). These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.     

In Vitro Acetylcholinesterase-Inhibitory Properties of Enzymatic Hemp Seed Protein Hydrolysates

The aim of this work was to characterize the structural and functional properties of hemp seed protein‐derived acetylcholinesterase (AChE)‐inhibitory enzymatic hydrolysates. Hemp seed protein isolate hydrolysis was performed using six different proteases (pepsin, papain, thermoase, flavourzyme, alcalase and pepsin + pancreatin) at different concentrations (1–4 %). The degree of hydrolysis was directly related to the amount of protease used but had no relationship with AChE‐inhibitory activity. Amino acid composition results showed that the hemp seed protein hydrolysates (HPHs) had high levels of negatively charged amino acids (39.62–40.18 %) as well as arginine. The 1 % pepsin HPH was the most active AChE inhibitor with ~6 µg/mL IC₅₀ value when compared to 8–11.6 µg/mL for the other HPHs. Mass spectrometry analysis showed that most of the peptides in all the hydrolysates were less than 1000 Da in size. However, the pepsin HPHs contained larger‐sized peptides (244–1009 Da) than the papain HPHs (246–758 Da), which in turn was larger than the alcalase HPH (246–607 Da). The higher AChE‐inhibitory effects of the pepsin HPHs may be due to increased synergistic effects from a wider peptide size range when compared to the papain and alcalase HPHs that had narrower ranges. The narrow peptide size range in the alcalase HPH confirms the higher efficiency of this protease in releasing small‐sized peptides from food proteins.     

Poly(propylene carbonate) polyurethane self‐polishing coating for marine antifouling application

In this contribution, a series of environmentally friendly thermoplastic poly(propylene carbonate) polyurethane (PPCU) were prepared by two‐step condensation polymerization and used to fabricate antifouling coatings. The poly(propylene carbonate) (PPC) segments served as degradable moieties. Quartz crystal microbalance with dissipation (QCM‐D) measurements revealed that the polyurethane could degrade in the presence of enzyme and the degradation rate increases with the decrease of the molecular weight of the polyurethane. Investigation on the hydrolytic degradation behavior and the release rate test of the antifoulants in artificial seawater also demonstrated that the hydrolysis rate and the release rate increased as the molecular weight decreased, which makes the coating controllable at the hydrolysis rate range of 0.012–0.051 g/(m²d). Marine field tests and algae settlement assay tests revealed that the polyurethane coating possessed antibiofouling ability due to its self‐renewal property and the release of antifoulants. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43667.     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

Investigation of Serine‐Proteinase‐Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI‐1)

Serine‐proteinase‐catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI‐1: both single peptides and two‐peptide chains (C‐ and N‐terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl–enzyme intermediate was preceded by hydrolysis of the substrate Lys–Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two‐peptide‐chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl–enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl–enzyme were not observed. The peptide splicing was sequence‐ not structure‐specific.