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Production and Purification of cutinase from Fusarium oxysporumUsing Modified Growth Media and a Specific cutinase Substrate

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Advances in Bioscience and Biotechnology 2015年第4期6卷 245-258页

作者： Ofir Degani Tel-Hai College Tel-Hai Israel Migal-Galilee Research Institute Kiryat Shmona Israel

cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. cutinase displayed hydrolytic activity not only towards cutin, the main component of the plant cuticle, but also towards a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Therefore, cutinase was evaluated for use in the chemical, food and cotton bio-scouring industry and for synthetic fibers modification. This research examined the production and purification of extracellular cutinase from the phytopathogenic fungus Fusarium oxysporum. The addition of apple cutin or its hydrolysate to the fungus growth medium resulted in an enhanced secretion of cutinase into the extracellular fluid. Testing 1-hexadecanol as an alternative to natural cutin to induce cutinase production resulted in a high process yield under modified growth conditions. cutinase enzyme production was followed by an optimized purification method for enzyme preparation using high-performance liquid chromatography and high-specificity 4-nitrophenyl (16-methyl sulfide ester) hexadecanoate (pNMSEH) cutinase substrate. Electrophoresis sodiumdodecyl sulfate-polyacrylamide and isoelectric focusing gels enabled the final separation and identification of the protein. The purified cutinase had an approximate molecular weight of 20 kDa and an isoelectric point of 4.7. The method presented here could be modified and used for cutinase production and purification in other microorganisms that exhibit cutinolytic activity.

关键词： 4-Nitrophenyl (16-Methyl Sulfide Ester) Hexadecanoate Substrate cutinase Enzymes Fusarium oxysporum Media Optimization

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Development of Medium and Kinetic Modeling for Enhanced Production of cutinase from Pseudomonas cepacia NRRL B-2320

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Advances in Microbiology 2013年第6期3卷 479-489页

作者： Kasturi Dutta Veeranki Venkata Dasu Krishnamoorthy Hegde Biochemical Engineering Laboratory Department of Biotechnology Indian Institute of Technology Guwahati Guwahati India

The development of medium for the production of cutinase from Pseudomonas cepacia NRRL B 2320 was carried out using Plackett-Burman experimental design followed by central composite design. The medium components were screened by Plackett-Burman experimental design which suggested that cutin, peptone, KCl and MgSO4·7H2O have influenced the cutinase production significantly with very high confidence levels. The concentration levels of these four components were optimized using 24 full factorial central composite design. An optimum combination of 10.06 g·L-1 of cutin, 17.77 g·L-1 of peptone, 0.635 g·L-1 of KCl and 5.455 g·L-1 of MgSO4·7H2O in the medium gave a maximum cutinase activity of 336 U·mL-1. An overall 2 fold increase in the production of cutinase was observed in the optimized medium. Growth and production of cutinase from P. cepacia NRRL B 2320 have been studied in shake flask and batch bioreactor. Time course of cell growth and enzyme production was fitted to the existing kinetic models reported in the literature to estimate the biokinetic parameters. These models suggested that the production of cutinase is growth associated in shake flask and it is a mixed growth type in a batch bioreactor.

关键词： cutinase Kinetics Bioprocessing Optimization Parameter Identification

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Expression, Purification and Crystallization of Thermostable Mutant of cutinase Est1 from Thermobifida alba

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Advances in Bioscience and Biotechnology 2018年第5期9卷 215-223页

作者： Kengo Kitadokoro Shingo Matsui Ryouhei Osokoshi Kensuke Nakata Shigeki Kamitani Departmentof Biomolecular Engineering Graduate School of Science and Technology Kyoto Institute of Technology Kyoto Japan Graduate School of Comprehensive Rehabilitation College of Health and Human Sciences Osaka Prefecture University Osaka Japan

A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 Åresolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 127.2 Å, b = 42.1 Å, c = 63.2 Å, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.

关键词： cutinase Themobifida alba Plastic Degrading Esterase Crystallization High Resolution Crystallographic Data

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Temperature and pH Dependent Deactivation of cutinases from Thermobifida fusca: A Comparative Study of Homologous Enzymes

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Modern Research in Catalysis 2014年第4期3卷 128-135页

作者： Krishnamoorthy Hegde Venkata Dasu Veeranki Biochemical Engineering Laboratory Department of Biotechnology Indian Institute of Technology Guwahati Guwahati India

Thermostability of two homologous cutinases, Cut1 and Cut2 from Thermobifida fusca NRRL B-8184 was inves-tigated at combination of different pH and temperature in the range of pH 6 - 9 and temperature 45℃ - 80℃, re-spectively. The deactivation rate constants, the half-life and thermodynamic parameters, viz., △H*, △S*, △G* and activation energy kinetics of inactivation of the cutinases were assessed at different combinations of pH and temperature and compared. The optimal pH and temperature for the least degree of deactivation for Cut1 and Cut2 were found to be 8℃ and 45℃, respectively. The deactivation process was found to be faster at pH 6 and 9, with minimum deactivation at pH 8 for both the cutinases. It was found that △S* values are negative for both the enzymes and △H* value of Cut2 was 1.5 fold higher than that of Cut1 in the range of pH studied. Cut2 was found to be thermodynamically more stable with 1.7 fold higher deactivation energy at pH 6 and 7 and 1.4 fold higher deactivation energy at pH 8 and 9 in comparison to Cut1.

关键词： cutinase Thermal Deactivation Thermostability Thermodynamics

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