Statistical Analysis of the Role of Cavity Flexibility in Thermostability of Proteins.

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, -0.6484 and -0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins.

Improving the catalytic performance of xylanase from Bacillus circulans through structure-based rational design.

Endo-1,4-ß-xylanase is one of the most important enzymes employed in biorefineries for obtaining fermentable sugars from hemicellulosic components. Herein, we aimed to improve the catalytic performance of Bacillus circulans xylanase (Bcx) using a structure-guided rational design. A systematic analysis of flexible motions revealed that the R49 component of Bcx (i) constrains the global conformational changes essential for substrate binding and (ii) is involved in modulating flexible motion. Site-saturated mutagenesis of the R49 residue led to the engineering of the active mutants with the trade-off between flexibility and rigidity. The most active mutant R49N improved the catalytic performance, including its catalytic efficiency (7.51-fold), conformational stability (0.7 °C improvement), and production of xylose oligomers (2.18-fold higher xylobiose and 1.72-fold higher xylotriose). The results discussed herein can be applied to enhance the catalytic performance of industrially important enzymes by controlling flexibility.

Improving the organic solvent resistance of lipase a from Bacillus subtilis in water-ethanol solvent through rational surface engineering.

Given that lipase is an enzyme applicable in various industrial fields and water-miscible organic solvents are important reaction media for developing industrial-scale biocatalysis, a structure-based strategy was explored to stabilize lipase A from Bacillus subtilis in a water-ethanol cosolvent. Site-directed mutagenesis of ethanol-interacting sites resulted in 4 mutants, i.e., Ser16Gly, Ala38Gly, Ala38Thr, and Leu108Asn, which were stable in 50% ethanol and had up to 1.8-fold higher stability than the wild-type. In addition, Leu108Asn was more thermostable at 45 °C than the wild type. The results discussed in this study not only provide insights into strategies for enzyme engineering to improve organic solvent resistance but also suggest perspectives on pioneering routes for constructing enzyme-based biorefineries to produce value-added fuels and chemicals.

Identification and classification of honey's authenticity by attenuated total reflectance Fourier-transform infrared spectroscopy and chemometric method.

BACKGROUND AND AIM: The authentication of honey is important to protect industry and consumers from such adulterated honey. However, until now, there has been no guarantee of honey's authenticity, especially in Indonesia. The classification of honey is based on the bee species (spp.) that produces it. The study used honey from sting bee Apis spp. and stingless bee Tetragonula spp. based on the fact that the content off honey produced between them has differences. Authenticating honey with currently available rapid detection methods, such as 13C nuclear magnetic resonance analysis, is costly. This study aimed to develop an inexpensive, fast, precise, and accurate classification method for authenticating honey. MATERIALS AND METHODS: In this study, we use attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy with wavelengths ranging between 550 and 4000 cm-1 as an alternative analysis method, which is relatively less expensive. The spectra of authentic and fake honey samples were obtained using ATR-FTIR and plotted using chemometric discriminant analysis. The authentic honey samples were acquired from a local Indonesian breeder of honey bees, while the fake honey samples were made from a mixture of water, sugar, sodium bicarbonate, and authentic honey. Data were collected using Thermo Scientific's OMNIC FTIR software and processed using Thermo Scientific's TQ Analyst software. RESULTS: Our method effectively classified the honey as authentic or fraudulent based on the FTIR spectra. To authenticate the honey, we formed two classes: Real honey and fake honey. The wavelengths that can best differentiate between these two classes correspond to four regions: 1600-1700 cm-1; 1175-1540 cm-1; 940-1175 cm-1; and 700-940 cm-1. Similarly, for classification purpose, we formed two classes: Apis spp. and Tetragonula spp. The wavelength region that can best classify the samples as belonging to the Apis spp. or Tetragonula spp. class is explicitly within the range of 1600-1700 cm-1. CONCLUSION: This study successfully demonstrated a method to rapidly and accurately classify and authenticate honey. ATR-FTIR is a useful tool to test the authenticity of honey.

A perspective on the biotechnological applications of the versatile tyrosinase.

Tyrosinase (E.C. 1.14.18. 1) is a type of Cu-containing oxidoreductase which has bifunctional activity for various phenolic substrates: ortho-hydroxylation of monophenols to diphenols (a cresolase activity) and oxidation of diphenols to quinones (a catecholase activity). Based on the broad substrate spectrum, tyrosinase has been used in bioremediation of phenolic pollutants, constructing biosensors for identifying phenolic compounds, and L-DOPA synthesis. Furthermore, not only tyrosinase has been used to produce useful polyphenol derivatives, but also it is recently revealed that the promiscuous activity of tyrosinase is closely related with delignification in the biorefinery. Accordingly, tyrosinase might be a potential biocatalyst for industrial applications (e.g., electroenzymatic L-DOPA production, but its long-term stability and reusability should be further explored. In this review, we emphasize the versatility of tyrosinase, which includes conventional applications, and suggest new perspectives as an industrial biocatalyst (e.g., electroenzymatic L-DOPA production). Especially, this review focuses on and comprehensively discusses recent innovative studies.

Engineering D-Lactate Dehydrogenase from Pediococcus acidilactici for Improved Activity on 2-Hydroxy Acids with Bulky C3 Functional Group.

Engineering D-lactic acid dehydrogenases for higher activity on various 2-oxo acids is important for the synthesis of 2-hydroxy acids that can be utilized in a wide range of industrial fields including the production of biopolymers, pharmaceuticals, and cosmetic compounds. Although there are many D-lactate dehydrogenases (D-LDH) available from a diverse range of sources, there is a lack of biocatalysts with high activities for 2-oxo acids with large functional group at C3. In this study, the D-LDH from Pediococcus acidilactici was rationally designed and further engineered by controlling the intermolecular interactions between substrates and the surrounding residues via analysis of the active site structure of D-LDH. As a result, Y51L mutant with the catalytic efficiency on phenylpyruvate of 2200 s-1 mM-1 and Y51F mutant on 2-oxobutryate and 3-methyl-2-oxobutyrate of 37.2 and 23.2 s-1 mM-1 were found, which were 138-, 8.5-, and 26-fold increases than the wild type on the substrates, respectively. Structural analysis revealed that the distance and the nature of the interactions between the side chain of residue 51 and the substrate C3 substituent group significantly affected the kinetic parameters. Bioconversion of phenyllactate as a practical example of production of the 2-hydroxy acids was investigated, and the Y51F mutant presented the highest productivity in in vitro conversion of D-PLA.

A novel d-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria: Structural analysis on the substrate specificity.

2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. d-lactate dehydrogenases (d-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the highest kcat/Km on PPA with 1,348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the d-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.

Perspectives for biocatalytic lignin utilization: cleaving 4-O-5 and Cα-Cß bonds in dimeric lignin model compounds catalyzed by a promiscuous activity of tyrosinase.

BACKGROUND: In the biorefinery utilizing lignocellulosic biomasses, lignin decomposition to value-added phenolic derivatives is a key issue, and recently biocatalytic delignification is emerging owing to its superior selectivity, low energy consumption, and unparalleled sustainability. However, besides heme-containing peroxidases and laccases, information about lignolytic biocatalysts is still limited till date. RESULTS: Herein, we report a promiscuous activity of tyrosinase which is closely associated with delignification requiring high redox potentials (>1.4 V vs. normal hydrogen electrode [NHE]). The promiscuous activity of tyrosinase not only oxidizes veratryl alcohol, a commonly used nonphenolic substrate for assaying ligninolytic activity, to veratraldehyde but also cleaves the 4-O-5 and Cα-Cß bonds in 4-phenoxyphenol and guaiacyl glycerol-ß-guaiacyl ether (GGE) that are dimeric lignin model compounds. Cyclic voltammograms additionally verified that the promiscuous activity oxidizes lignin-related high redox potential substrates. CONCLUSION: These results might be applicable for extending the versatility of tyrosinase toward biocatalytic delignification as well as suggesting a new perspective for sustainable lignin utilization. Furthermore, the results provide insight for exploring the previously unknown promiscuous activities of biocatalysts much more diverse than ever thought before, thereby innovatively expanding the applicable area of biocatalysis.

Enhanced solubility of piperine using hydrophilic carrier-based potent solid dispersion systems.

CONTEXT: Piperine alkaloid, an important constituent of black pepper, exhibits numerous therapeutic properties, whereas its usage as a drug is limited due to its poor solubility in aqueous medium, which leads to poor bioavailability. OBJECTIVE: Herein, a new method has been developed to improve the solubility of this drug based on the development of solid dispersions with improved dissolution rate using hydrophilic carriers such as sorbitol (Sor), polyethylene glycol (PEG) and polyvinyl pyrrolidone K30 (PVP) by solvent method. Physical mixtures of piperine and carriers were also prepared for comparison. METHODS: The physicochemical properties of the prepared solid dispersions were examined using SEM, TEM, DSC, XRD and FT-IR. In vitro dissolution profile of the solid dispersions was recorded and compared with that of the pure piperine and physical mixtures. The effect of these carriers on the aqueous solubility of piperine has been investigated. RESULTS: The solid dispersions of piperine with Sor, PEG and PVP exhibited superior performance for the dissolution of piperine with a drug release of 70%, 76% and 89%, respectively after 2 h compared to physical mixtures and pure piperine, which could be due to its transformation from crystalline to amorphous form as well as the attachment of hydrophilic carriers to the surface of poorly water-soluble piperine. CONCLUSION: Results suggest that the piperine solid dispersions prepared with improved in vitro release exhibit potential advantage in delivering poorly water-soluble piperine as an oral supplement.

Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration.

Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.

Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

Engineering substrate specificity of succinic semialdehyde reductase (AKR7A5) for efficient conversion of levulinic acid to 4-hydroxyvaleric acid.

Engineering enzyme substrate specificity is a promising approach that can expand the applicability of enzymes for the biocatalytic production of industrial chemicals and fuels. In this study, succinic semialdehyde reductase (AKR7A5) was engineered for the conversion of levulinic acid to 4-hydroxyvaleric acid. Levulinic acid is a derivative of cellulosic biomass, and 4-hydroxyvaleric acid is a potential precursor to bio-polymers and fuels. Therefore, the enzymatic conversion of levulinic acid to 4-hydroxyvaleric acid is of special significance in that this conversion could provide a meaningful basis for the bio-production of useful chemicals from cellulosic biomass. In engineering the substrate specificity of the AKR7A5, a rational design approach with the aid of enzyme-substrate interatomic contact analysis was applied. The Met13 residue was selected as a key mutation site, and substitutions of the residue with six hydrophobic amino acids were applied. As a result, four mutants with enhanced catalytic activity toward levulinic acid were obtained, and the most improved mutant, Met13Trp, exhibited a 7.0-fold increase in catalytic efficiency. Additionally, the structural effects of the positive mutations were investigated to analyze the structural basis for the enzyme substrate specificity with the target substrate.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.

Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

Computational approach for designing thermostable Candida antarctica lipase B by molecular dynamics simulation.

Candida antarctica lipase B (CalB) is one of the most useful enzyme for various reactions and bioconversions. Enhancing thermostability of CalB is required for industrial applications. In this study, we propose a computational design strategy to improve the thermostability of CalB. Molecular dynamics simulations at various temperatures were used to investigate the common fluctuation sites in CalB, which are considered to be thermally weak points. The RosettaDesign algorithm was used to design the selected residues. The redesigned CalB was simulated to verify both the enhancement of intramolecular interactions and the lowering of the overall root-mean-square deviation (RMSD) values. The A251E mutant designed using this strategy showed a 2.5-fold higher thermostability than the wild-type CalB. This strategy could apply to other industry applicable enzymes.

Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack.

Despite the potential use of peroxidases as industrial biocatalysts, their practical application is often impeded due to suicide inactivation by radicals generated in oxidative reactions. Using a peroxidase from Coprinus cinereus (CiP) as a model enzyme, we revealed a dominant factor for peroxidase inactivation during phenol oxidation, and we engineered radical-stable mutants by site-directed mutagenesis of an amino acid residue susceptible to modification by phenoxyl radical. Mass spectrometry analysis of inactivated CiP identified an adduct between F230 and a phenoxyl radical, and subsequently, the F230 residue was mutated to amino acids that resisted radical coupling. Of the F230 mutants, the F230A mutant showed the highest stability against radical inactivation, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. The F230A mutant also exhibited a 16-fold higher turnover of the phenol substrate compared with the wild-type enzyme. Furthermore, the F230A mutant was stable during the oxidation of other phenolic compounds, including m-cresol and 3-methoxyphenol. No structural changes were observed by UV-vis and CD spectra of CiP after radical coupling, implying that the F230-phenol radical adduct inactivated CiP by blocking substrate access to the active site. Our novel strategy can be used to improve the stability of other peroxidases inactivated by radicals.

Enhancing the activity of Bacillus circulans xylanase by modulating the flexibility of the hinge region.

Enzymes undergo multiple conformational changes in solution, and these dynamics are considered to play a critical role in enzyme activity. Hinge-bending motions, resulting from reciprocal movements of dynamical quasi-rigid bodies, are thought to be related to turnover rate and are affected by the physical properties of the hinge regions. In this study, hinge identification and flexibility modification of the regions by mutagenesis were conducted to explore the relationship between hinge flexibility and catalytic activity. Bacillus circulans xylanase was selected for the identification and mutation of the hinge regions. As a result, turnover rate (V(max)) was improved approximately twofold in mutants that have more rigid hinge structure, despite the decrease in K(m) and V(max)/K(m). This result indicates that the rigidly mutated hinge has positive effects on B. circulans xylanase activity.

High-rate partial nitritation using porous poly(vinyl alcohol) sponge.

Poly(vinyl alcohol) (PVA) has been utilized as a support material for the immobilization of nitrifying bacteria without the comprehensive survey of partial nitritation. In the present study, the activities of nitrifiers and the maximum nitrogen conversion rate of partial nitritation with PVA sponge-cubes were specified according to different conditions. The selective enrichment of ammonia-oxidizing bacteria (AOB) on PVA sponge-cubes was achieved by the competition between AOB and nitrite-oxidizing bacteria for dissolved oxygen. The efficiency of ammonia oxidation was proportional to the concentration of HCO3 (-) with the molar ratio of HCO3 (-)-C/NH4 (+)-N = 1.91 and a half of the ratio was applied to the further experiments to ensure stable partial nitritation. The maximum nitrogen conversion rate of partial nitritation was dependent on the volume, not the size of sponge-cubes. The partial nitritation showed the superior rate performance of 3.09 kg N/m(3) day with the packing ratio of 32 % of 5 × 5 × 5 mm(3) PVA sponge-cubes.