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Jian Chen - One of the best experts on this subject based on the ideXlab platform.
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extracellular location of thermobifida fusca cutinase expressed in escherichia coli bl21 de3 without mediation of a signal peptide
Applied and Environmental Microbiology, 2013Co-Authors: Ronald W Woodard, Jian ChenAbstract:Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinaseNS) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinaseNS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinaseNSS130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinaseNSS130A, the cells expressing cutinaseNS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of “cell leakage induced by the limited phospholipid hydrolysis of cutinaseNS” was proposed to explain the underlying mechanism for the extracellular release of cutinaseNS.
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characterization of thermobifida fusca cutinase carbohydrate binding module fusion proteins and their potential application in bioscouring
Applied and Environmental Microbiology, 2010Co-Authors: Yao Zhang, Sheng Chen, Artur Cavocopaulo, Jian ChenAbstract:Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBMCel6A and cutinase-CBMCenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50°C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBMCel6A and by 28% for cutinase-CBMCenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.
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Biochemical characterization of the Cutinases from Thermobifida fusca
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Sheng Chen, Susan Billig, Wolfgang Zimmermann, Jian ChenAbstract:Abstract Thermobifida fusca produces two Cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca Cutinases for the first time. For a better comparison between bacterial and fungal Cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal Cutinases are monomeric proteins in solution. The bacterial Cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca Cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca Cutinases, they may have promising applications in related industries.
Richard A. Gross - One of the best experts on this subject based on the ideXlab platform.
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immobilized Cutinases preparation solvent tolerance and thermal stability
Enzyme and Microbial Technology, 2018Co-Authors: Abhijit N. Shirke, Joshua Baik, Yi Zou, Richard A. GrossAbstract:Abstract Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized Cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields >98% were achieved for all three Cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 °C for 24 h. Among the three Cutinases, HiC has the highest tolerance towards solvents of increased polarity while TtC has the highest thermal stability (up to 80 °C) in a bulk reaction system that consists of the reactants butanol and lauric acid. In nonane, these Cutinases retain >64% of their activity at 90 °C. Furthermore, kinetic stability (residual activity as a function of time) analysis reveals that the Cutinases retain >75% residual activity at 70 °C in 3 h. Moreover, at 80 °C, the kinetic stability of TtC is higher than that of HiC and AoC. Collectively, the results herein set the stage for the in-depth evaluation of these catalysts for selective transformations in organic media.
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Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis
2018Co-Authors: Abhijit N. Shirke, Glenn L. Butterfoss, Christine White, Jacob A. Englaender, Allison Zwarycz, Robert J. Linhardt, Richard A. GrossAbstract: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
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Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.
Biotechnology and bioengineering, 2016Co-Authors: Abhijit N. Shirke, J. Andrew Jones, Glenn L. Butterfoss, Mattheos A. G. Koffas, Jin Ryoun Kim, Richard A. GrossAbstract: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.
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identification and comparison of Cutinases for synthetic polyester degradation
Applied Microbiology and Biotechnology, 2012Co-Authors: P J Baker, Richard A. Gross, Christopher S Poultney, Zhiqiang Liu, Jin Kim MontclareAbstract:Cutinases have been exploited for a broad range of reactions, from hydrolysis of soluble and insoluble esters to polymer synthesis. To further expand the biotechnological applications of Cutinases for synthetic polyester degradation, we perform a comparative activity and stability analysis of five Cutinases from Alternaria brassicicola (AbC), Aspergillus fumigatus (AfC), Aspergillus oryzae (AoC), Humicola insolens (HiC), and the well-characterized Fusarium solani (FsC). Of the Cutinases, HiC demonstrated enhanced poly(e-caprolactone) hydrolysis at high temperatures and under all pH values, followed by AoC and AfC. Both AbC and FsC are least stable and function poorly at high temperatures as well as at acidic pH conditions. Surface charge calculations and phylogenetic analysis reveal two important modes of cutinase stabilization: (1) an overall neutral surface charge within the “crowning area” by the active site and (2) additional disulfide bond formation. These studies provide insights useful for reengineering such enzymes with improved function and stability for a wide range of biotransformations.
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structural and functional studies of aspergillus oryzae cutinase enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation
Journal of the American Chemical Society, 2009Co-Authors: Yuying Gosser, Glenn L. Butterfoss, Richard A. Gross, P J Baker, Yaniv Ravee, Ziying Lu, Girum Alemu, Huiguang Li, Xiangpeng Kong, Jin Kim MontclareAbstract:Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.
Doris Ribitsch - One of the best experts on this subject based on the ideXlab platform.
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small cause large effect structural characterization of Cutinases from thermobifida cellulosilytica
Biotechnology and Bioengineering, 2017Co-Authors: Doris Ribitsch, Alessandro Pellis, Altijana Hromic, Sabine Zitzenbacher, Barbara Zartl, Caroline Gamerith, Alois Jungbauer, Andrzej łyskowski, Georg Steinkellner, Karl GruberAbstract:We have investigated the structures of two native Cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different Cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four Cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.
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Two novel class II hydrophobins from Trichoderma spp. stimulate enzymatic hydrolysis of poly(ethylene terephthalate) when expressed as fusion proteins.
Applied and Environmental Microbiology, 2013Co-Authors: Liliana Espino-rammer, Agnieszka Przylucka, Annemarie Marold, Doris Ribitsch, Katrin Greimel, Enrique Herrero Acero, Georg M Guebitz, Christian P Kubicek, Irina S DruzhininaAbstract:Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial Cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of Cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.
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enzymatic surface hydrolysis of pet effect of structural diversity on kinetic properties of Cutinases from thermobifida
Macromolecules, 2011Co-Authors: Enrique Herrero Acero, Doris Ribitsch, Katrin Greimel, Wolfgang Zimmermann, Ren Wei, Georg Steinkellner, Karl Gruber, Inge Eiteljoerg, Eva Trotscha, Manfred ZinnAbstract:In this study Cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two Cutinases from T. fusca KW3. The three isolated Thermobifida Cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water co...
Artur Cavacopaulo - One of the best experts on this subject based on the ideXlab platform.
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production of heterologous Cutinases by e coli and improved enzyme formulation for application on plastic degradation
Electronic Journal of Biotechnology, 2013Co-Authors: Daniela S Gomes, Teresa Matama, Artur Cavacopaulo, Galba Maria De Campostakaki, Alexandra A SalgueiroAbstract:Background: The hydrolytic action of Cutinases has been applied to the degradation of plastics. Polyethylene terephthalate (PET) have long half-life which constitutes a major problem for their treatment as urban solid residues. The aim of this work was to characterize and to improve stable the enzyme to optimize the process of degradation using enzymatic hydrolysis of PET by recombinant Cutinases. Results: The wild type form of cutinase from Fusarium solani pisi and its C-terminal fusion to cellulose binding domain N1 from Cellulomonas fimi were produced by genetically modified Escherichia coli . The maximum activity of Cutinases produced in Lactose Broth in the presence of ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) was 1.4 IU/mL. Both Cutinases had an optimum pH around 7.0 and they were stable between 30 and 50oC during 90 min. The addition of glycerol, PEG-200 and (NH 4 ) 2 SO 4 to the metabolic liquid, concentrated by ultra filtration, stabilized the activity during 60 days at 28oC. The treatment of PET with Cutinases during 48 hrs led to maxima weight loss of 0.90%. Conclusions: Recombinant microbial Cutinases may present advantages in the treatment of poly(ethylene terephthalate) PET through enzymatic treatments.
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direct enzymatic esterification of cotton and avicel with wild type and engineered Cutinases
Cellulose, 2013Co-Authors: Teresa Matama, Margarida Casal, Artur CavacopauloAbstract:In this work, the surface of cellulose, either Avicel or cotton fabric, was modified using Cutinases without any previous treatment to swell or to solubilise the polymer. Aiming further improvement of cutinase ester synthase activity on cellulose, an engineered cutinase was investigated. Wild-type cutinase from Fusarium solani and its fusion with the carbohydrate-binding module N1 from Cellulomonas fimi were able to esterify the hydroxyl groups of cellulose with distinct efficiencies depending on the acid substrate/solvent system used, as shown by titration and by ATR-FTIR. The carbonyl stretching peak area increased significantly after enzymatic treatment during 72 h at 30 °C. Cutinase treatment resulted in relative increases of 31 and 9 % when octanoic acid and vegetable oil were used as substrates, respectively. Cutinase-N1 treatment resulted in relative increases of 11 and 29 % in the peak area when octanoic acid and vegetable oil were used as substrates, respectively. The production and application of cutinase fused with the domain N1 as a cellulose ester synthase, here reported for the first time, is therefore an interesting strategy to pursuit.
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hydrolysis of cutin by pet hydrolases
Macromolecular Symposia, 2010Co-Authors: Justyna Teresa Korpecka, Artur Cavacopaulo, Susan Billig, Wolfgang Zimmermann, Sonja Heumann, Manfred Zinn, Julian Ihssen, Georg M GuebitzAbstract:Functionalisation of synthetic polymers by using enzymes has been recently demonstrated. The major advantage of enzymes over chemical processes lies in their surface specific and endo-wise mode of action. Surface hydrophilisation of PET with lipases and Cutinases leads to a dramatic increase of the surfacial acid and hydroxyl group content while conventional chemical treatment does not cause any change. However, this PET-hydrolysing activity by enzymes from distinct classes has not yet been correlated to activity on natural polyesters. Here, we show that lipases, Cutinases and a PHA-depolymerase are all capable of hydrolysing PET, while only lipases and Cutinases also hydrolysed cutin to various degrees. Lipases showed a higher specificity for terminal fatty acids while the Cutinases preferred hydroxy fatty acids during cutin hydrolysis.
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enzymatic surface hydrolysis of poly ethylene terephthalate and bis benzoyloxyethyl terephthalate by lipase and cutinase in the presence of surface active molecules
Journal of Biotechnology, 2009Co-Authors: Anita Eberl, Artur Cavacopaulo, Sonja Heumann, Tina Bruckner, Rita Araujo, Franz Kaufmann, Wolfgang Kroutil, Georg M GuebitzAbstract:A lipase from Thermomyces lanuginosus and Cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.
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effect of the agitation on the adsorption and hydrolytic efficiency of Cutinases on polyethylene terephthalate fibres
Enzyme and Microbial Technology, 2007Co-Authors: Alexandre Oneill, Margarida Casal, Georg M Guebitz, Rita Araujo, Artur CavacopauloAbstract:The effect of agitation on adsorption, desorption and hydrolytic efficiency of a native and a genetically modified cutinase (L182A) on polyethylene terephthalate fibres is reported in this paper. The effect of mechanical agitation was studied using a shaker bath with orbital agitation and a Rotawash machine with vertical agitation with and without extra steel discs inside the reaction pots. The results obtained indicate that mechanical agitation combined with enzymatic action enhances the adsorption and activity of Cutinases towards PET (polyethylene terephthalate) fibres. L182A showed higher adsorption than the native enzyme for all the levels of mechanical agitation. Lower units of L182A lead to similar yields of terephthalic acid formed in all levels of mechanical agitation. The highest increase of hydroxyl surface groups was found for the genetically modified L182A at the lowest level of mechanical agitation with a shaker bath. These results indicate that enzymatic functionalization of PET is favoured with a process with lower levels of mechanical agitation.
Ruoyu Hong - One of the best experts on this subject based on the ideXlab platform.
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Cutinases catalyze polyacrylate hydrolysis and prevent their aggregation
Polymer Degradation and Stability, 2019Co-Authors: Ruoyu HongAbstract:Abstract During the recycling of waste paper, the accumulation of polyacrylates present in the waste paper causes the formation of tacky substances known as stickies. Deposition of these stickies on the machinery decreases the quality of the recycled paper and increases the usage of circulating water. Enzymes that hydrolyze these polyacrylates can minimize or eliminate the deposition of stickies. In initial experiments, the abilities of the Cutinases from Humicola insolens, Fusarium solani and Thermobifida fusca to hydrolyze poly (methyl acrylate) (PMA) and poly (ethyl acrylate) (PEA) within a macroporous resin were compared. Then, to simulate the environment encountered during paper recycling, PMA and PEA dispersions were used as substrates. The decrease in turbidity was measured at a concentration of 0.5 mg mL−1. When used at pH 8.0 and 30–50 °C, T. fusca cutinase limited the turbidity decrease to about 1.0% and favored the hydrolysis of PEA over PMA. F. solani and H. insolens Cutinases performed best at pH 8.5 and temperatures of 35 and 50 °C, respectively. At a polyacrylate concentration of 0.1 mg mL−1, the optimal temperatures of these Cutinases decreased. The optimal T. fusca cutinase dosage was lower than those of F. solani and H. insolens Cutinases.
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comparison of Cutinases in enzymic deinking of old newsprint
Cellulose, 2017Co-Authors: Ruoyu Hong, Lingqia Su, Zhu Long, Sheng Chen, Jing WuAbstract:In this study, the impact of Cutinases from Thermobifida fusca and Fusarium solani pisi on the deinking of old newsprint were evaluated for the first time. When repulped old newsprint was treated with 8 U/g of T. fusca cutinase at pH 8 and 60 $$^{\circ }$$ C for 30 min, the brightness of the deinked papers reached 42.01%. With 8 U/g F. solani cutinase at pH 8.5 and 35 $$^{\circ }$$ C for 30 min, the brightness reached 41.62%. These brightness values are higher than that achieved using chemical deinking by 5.13 and 4.38%, respectively. These brightness values were also superior to that achieved using commercial lipase in this study and those previously reported using cellulases, hemicellulases, and laccase, which were higher than that achieved using chemical deinking by 2–4%. The mechanical properties of the deinked paper, including tensile index, tear index, and burst index, were also measured. The results showed that the two Cutinases had different effects on the paper fibers. In summary, both T. fusca and F. solani pisi Cutinases were able to remove ink from old newsprint more efficiently than alkaline chemistry or other enzymes. This study provides a potential strategy to deink efficiently old newsprint using cutinase.
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enhanced extracellular expression of gene optimized thermobifida fusca cutinase in escherichia coli by optimization of induction strategy
Process Biochemistry, 2015Co-Authors: Ruoyu HongAbstract:Abstract Our previous study demonstrated that when Thermobifida fusca cutinase was expressed in Escherichia coli without mediation of a signal peptide, it could release to the culture medium via the enhanced membrane permeability, which was based on its limited phospholipid hydrolysis activity. The goal of the present work was to achieve highly efficient extracellular production of the recombinant signal peptide-free cutinase in E. coli . The codons of the T. fusca cutinase gene were optimized for expression in E. coli , and a recombinant expression system was constructed using this optimized gene. After that, the induction strategy was optimized using high-cell-density cultivation in a 3-L fermentor. Results showed that the optimal induction condition was at a dry cell weight (DCW) of 13 g L −1 , and an IPTG/lactose combination induction strategy is proposed, in which IPTG is added once in a final concentration of 25.0 μM, and lactose is fed at a rate of 0.5 g L −1 h −1 . In this condition, an extracellular cutinase activity of 2258.5 U mL −1 (5.1 g L −1 ) was achieved, which represented the highest cutinase production ever reported, and demonstrated the potential of this system for the industrial production of cutinase.
