Application of cutinase in the degradation of biodegradable polyester poly(butylene adipate-co-terephthalate) / 生物工程学报

With the environmental pollution caused by waste plastics becoming increasingly serious, biodegradable polyester has become the focus of public attention. Poly(butylene adipate-co-terephthalate) (PBAT) is a biodegradable polyester formed by the copolymerization of aliphatic and aromatic groups, which has excellent performance of both. The degradation of PBAT under natural conditions requires strict environmental conditions and long degradation cycle. To address these shortcomings, this study explored the application of cutinase in PBAT degradation and the impact of butylene terephthalate (BT) content on the biodegradability of PBAT, so as to improve the degradation rate of PBAT. Five Polyester degrading enzymes from different sources were selected to degrade PBAT to pick out the most efficient enzyme. Subsequently, the degradation rate of PBAT materials with different BT content were determined and compared. The results showed that cutinase ICCG was the best enzyme for PBAT biodegradation, and the higher the BT content, the lower the degradation rate of PBAT. Furthermore, the optimum temperature, buffer type, pH, the ratio of enzyme to substrate (E/S) and substrate concentration in the degradation system were determined to be 75 ℃, Tris HCl, 9.0, 0.4% and 1.0% respectively. These findings may facilitate the application of cutinase in PBAT degradation.

Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum / 生物工程学报

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.

The application of carbohydrate binding module-Thermobifida fusca cutinase fusion protein in polyethylene terephthalate degradation / 生物工程学报

With the development of global economy, the dramatically increased production of polyethylene terephthalate (PET) plastics has led to a remarkably increased amount of plastic waste. PET waste can be treated by landfill, incineration, or biodegradation. While landfilling and incineration may cause secondary pollution, biodegradation has since received increased attentions due to its environmental friendliness. Recent studies have indicated that the carbohydrate binding module (CBM) can effectively enhance the binding of PET degrading enzymes to PET, and consequently increasing PET degradation rate. Here we constructed a fusion protein BaCBM2-Tfuc containing the BaCBM2 from Bacillus anthraci and the cutinase Tfuc from Thermobifida fusca, by megaprimer PCR of whole plasmids (MEGAWHOP). Notabaly, the PET film degradation efficiency (at 60 ℃) of BaCBM2-Tfuc was 2.8 times that of Tfuc. This study may provide technical support for constructing fusion proteins capable of efficiently degrading PET.

Characterization of Humicola insolens cutinase-tachystatin A2 fusion protein and its application in treatment of recycled paper stickies / 生物工程学报

With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.

Studies on Immobilization of Cutinases from Thermobifida fusca on Glutaraldehyde Activated Chitosan Beads.

Aims: To evaluate and optimize the activity and stability performance of two recombinant cutinases of Thermobifida fusca, Cut1 and Cut2 on glutaraldehyde activated chitosan beads. Place and Duration of Study: Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiment conducted as a partial fulfillment to PhD degree from December, 2010 to January, 2014. Methodology: Purified cutinase were immobilized on chitosan beads by covalently coupling with glutaraldehyde. The biophysical properties of immobilized cutinase was analysed by FTIR, FESEM and the operational stability and activity of the immobilized cutinase was studied at different pH and temperature. Results: The optimal immobilization was achieved with 3% (v/v) glutaraldehyde activation and coupling pH of 8.5. Under this condition, 74% and 71% of immobilization was achieved for Cut1 and Cut2, respectively. Immobilized cutinase showed optimal activity at pH 8 with optimal functional range of pH 7.5 to 9 and 55ºC with operational stability in the range of 45ºC to 70ºC. The reusability and storage stability was found to be 80% after 10 reuse cycles and 50% after 13 days, respectively as compared to its initial activity. There was no loss in activity even after repeated freeze drying. Conclusion: the cutinase immobilized on glutaraldehyde activated chitosan beads demonstrated better operational stability in comparison to free cutinase, showing chitosan as a potential support in the enzyme immobilization technology for industrial applications of T. fusca cutinases.

Production of heterologous cutinases by E. coli and improved enzyme formulation for application on plastic degradation

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 (NH4)2SO4 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.

Inoculum padronization for the production of cutinase by Fusarium oxysporum

Cutinase is a versatile enzyme showing several interesting properties for application in industrial processes. The widespread use of this enzyme depends on the development of an efficient and low-cost production system. One of the most important steps in a fermentation process is the standardization of the inoculum characteristics. In this study, the production of cutinase by Fusarium oxysporum showed a statistically significant relationship with both the inoculum size and the inoculum PDA pH. The greatest activities were 19.1 U/mL at PDA pH 7.0 and 22.72 U/mL using an aliquot of 12.72 x 10(7) spores/mL. The macroscopic characteristics of the colonies of Fusarium oxysporum changed according to the variation of the medium pH, with the best results recorded in those colonies presenting a cotton white aspect.

Cutinase é uma enzima versátil, que apresenta propriedades interessantes para aplicação em processos industriais. O uso desta enzima em larga escala depende do desenvolvimento de um sistema de produção eficiente e de baixo custo. Uma das etapas mais importantes em um processo de fermentação é a padronização do inóculo. Neste estudo, houve uma associação estatisticamente significativa entre a produção de cutinase por Fusarium oxysporum e tamanho do inóculo e pH do meio PDA. As maiores atividades de cutinase foram 19,1 U/mL em PDA com pH 7,0 e 22,72 U/mL empregando um inóculo de 12,72 x 10(7) esporos/mL. As características macroscópicas das colônias de Fusarium oxysporum mostraram alterações em função do pH do meio, com as maiores atividades sendo registradas em presença de colônias brancas com aspecto cotonoso.