Jing Wu
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China.

Zheng Fei Yan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China.

ISBN 978-93-5547-303-5 (Print)
ISBN 978-93-5547-304-2 (eBook)
DOI: 10.9734/bpi/mono/978-93-5547-303-5

Cutinases, also known as cutin hydrolases (EC, first discovered from phytopathogenic fungi that grow on cutin as the sole carbon source. Cutin is a complex biopolymer composed of epoxy and hydroxy fatty acids and forms the structural component of higher plants cuticle. Cutinases are serine esterases with a classical Ser-His-Asp catalytic triad that belong to the \(\alpha\)/\(\beta\) hydrolase superfamily. It was found to be distinct among the \(\alpha\)/\(\beta\) hydrolases, unlike the majority of lipases and esterases, which is able to hydrolyse lipid substrates and, its activity is not activated by interfacial effects-it efficiently hydrolyzes hydrophobic substrates in solution or in emulsions.

Cutinases were identified in the 1960's and characterized in the early 1970’s. The cutinase from the filamentous fungus Fusarium solani pisi rapidly became a model system for the study of cutinase structure, function and reactivity. There are few studies on bacterial cutinases because of the lack of open reading frames. This book begins by presenting recent progress concerning the detail information of Thermobifida fusca cutinase as one of bacterial cutinases, highlighting the structural characteristics and catalytic activities that distinguish cutinases from related enzymes. Its versatile catalytic abilities led to it was potentially useful for a variety of industrial applications.

In this book, four sections (Total 11 chapters) are organized by our groups, which summarizes identification, modification, preparation and application of T. fusca cutinase. Section 1 (chapter 1 and 2) that edited by Ling Qia Su and Sheng Chen reviews identification and characterization of cutinase. Section 2 (chapter 3 and 4) that edited by Wei Xia and Kang Zhang focuses on structure activity relationship and molecular modification of cutinase. Section 3 (chapter 5, 6 and 7) that edited by Ling Qia Su, Zheng Fei Yan, Lei Wang and Yan Huang introduces preparation of cutinase by overexpression and its fermentation strategy. Section 4 (chapter 8, 9, 10 and 11) that edited by Zhan Zhi Liu, Zheng Fei Yan and Wei Xia enumerates some cases about application of cutinase as natural green friendly methodology in textile, papermaking and ester synthesis industry.

Special thanks are due to our group, without whose constant efforts the volumes could not be published. We believe that we have tried our best to draw a more comprehensive atlas for the development of cutinase. We also wish to thank everyone involved in making this book possible and hope that it will become a tool equally useful to researchers, industrialists and students.


Cutinase: Preparation and Application

Jing Wu, Zheng Fei Yan

Cutinase: Preparation and Application, 27 November 2021, Page 1

The book summarizes the preparation and application of baterial cutinase, which offers the author a chance to comprehensive understanding of cutinase and lays the foundations for application of cutinase in the future. Firstly, his book provides the systemic information of Thermobifida fusca cutinase, such as concept, source, identification, phylogenetic evolution, and crystal structure. Secondly, its functional activity of cutinase was investigated and increased by molecular modification. Thirdly, the large-scale preparation of cutinase was introduced by overexpression and fermentation strategy. Lastly, the book enumerates some cases about application of cutinase as natural green friendly methodology in textile, papermaking and ester synthesis industry. The book is written for researchers, professional/practioners and graduate students in the field of cell biology, molecular biology and biophysics.

Identification and Distribution of Cutinase

Ling Qia Su

Cutinase: Preparation and Application, 27 November 2021, Page 2-10

Cutinases are present in bacteria and fungi, which exhibit a hydrolytic capacity for the ester bonds in cutin. Currently, cutinases from fungi were extensively investigated. So far, there are few studies on cutinases from bacteria because of the lack of open reading frames. In this study, cutinase was secreted from Thermobifida fusca that induced by cutin, which was purified with the p-nitrophenyl butyrate hydrolyzing activity. Mass fingerprinting analysis showed that the protein band matched two proteins, Tfu_0883, and Tfu_0882, which have 93% similarity with both sequences. Then, these genes were cloned and overexpressed in Escherichia coli, which were identified as cutinases by their capability to hydrolyze cutin. This is the first report of cutinase encoding genes from bacterial source. Also, the bacterial and fungal enzymes are demonstrated no sequence similarity, which was classified into prokaryotic and eukaryotic cutinase subfamilies, respectively.

Characterization of Cutinase

Sheng Chen

Cutinase: Preparation and Application, 27 November 2021, Page 11-19

In previous work, T. fusca cutinases (Tfu_0883 and Tfu_0882) have been isolated and identified, with 93% similarity in amino acid sequence. Biochemical characteristics of T. fusca cutinases were investigated for the first time. F. solani pisi cutinase was used as a control for a better comparison of cutinases from bacterium and fungi. This study has shown that T. fusca cutinases as monomeric proteins exhibited a broad substrate specificity, which could hydrolyze plant cutin, insoluble triglycerides, and soluble esters. T. fusca and F. solani pisi cutinase are similar in substrate kinetics without interfacial activation. T. fusca cutinases showed higher thermal stability in the presence of surfactants and organic solvents, which might have promising applications in related industries.

Structure-function Studies of Cutinase

Wei Xia

Cutinase: Preparation and Application, 27 November 2021, Page 20-39

T. fusca cutinase has been cloned and expressed in Escherichia coli, which is capable of degrading not only plant cutin, but also a variety of soluble synthetic esters, and insoluble triglycerides. So far, there is no report about the crystal structure of T. fusca cutinase. Thus, in this study, high-quality crystals have been obtained from purified T. fusca cutinase protein for the first time and collected a set of reflection data at 1.54 Å. The final crystal structure was confirmed and obtained, whose R factor and free_R factor are 20.8% and 21.6% respectively. The catalytic center is shown as Ser130-His208-Asp176, the oxyanion hole is located at Try60-Met131, and the substrate site is occupied by H2O molecule. And then, structural comparison between T. fusca cutinase and other cutinase was also investigated. Based on structural analysis, the disulfide bond is the key core for the thermal stability of T. fusca cutinase. To investigate its contribution, cutinase mutant without disulfide bond (C241A/C259A) was constructed and expressed in Escherichia coli. The extracellular yield production of mutant decreased dramatically to 13.8%, and most was detected in cell as insoluble inclusion bodies. Its catalytic efficiency also decreased to 71.0%. Besides, CD spectra analysis showed that the secondary structure of mutant changed distinctly, which lead to reducing the thermal stability.

Modification of Cutinase

Kang Zhang

Cutinase: Preparation and Application, 27 November 2021, Page 40-57

Based on the crystal structure of T. fusca cutinase (Tfu_0883), 17 amino acid sites that were selected might influence the binding affinity of cutinase to cutin or its ability to recruit the substrate. Among them, 7 mutants exhibited higher hydrolysis activity by using \(\rho\)-nitrophenyl butyrate (\(\rho\)NPB) as substrate, in comparison to that of wild-type. L90A and I213A mutants were discovered to have better hydrolysis ability of tomato cuticle cutin, about 5 and 2.4 times of wild-type, respectively. Molecular dynamics (MD) showed that in L90A, the conformation of the loop 58-64, especially Tyr60 was flipped noticeably towards Ala90, leaving more space for substrate binding. Similarly, the loop 174-182 and F209 in I213A was also found flipped towards the solvent environment, enlarging the substrate binding space, increasing the binding affinity, and eventually facilitating the catalytic process. In addition, T. fusca cutinase that fused with the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBMCel6A) or Cellulomonas fimi cellulase CenA (CBMCenA) also increased binding activity. Fusion enzymes displayed similar catalytic properties and pH stabilities to that of T. fusca cutinase. Its half-life was 53 h at the optimal temperature of 50\(^{\circ}\)C. Meanwhile, the adsorption effect of fusion enzymes was also investigated. Results revealed that the adsorption rates of cutinase-CBMCel6A and cutinase-CBMCenA on filter paper reached 63% and 64%, which were 51% and 52% higher than that of native cutinase, respectively, suggesting that better applications in textile bioscouring than native cutinase.

T. fusca cutinase was extracellularly expressed by the mediation of pelB signal peptide via the Type II secretion pathway. A translational initial region degeneracy mutagenesis was carried out in the initial sequence of pelB. A fast screening method for these mutants was developed and a high cutinase production level achieved 38.0 U/mL. In addition, recombinant Escherichia coli that expressed precursor cutinase with one and two cleavage sites in signal peptide were constructed. The cutinase activity in culture medium was 95.7 U/mL and 130.3 U/mL, respectively, indicating that the increase of signal peptide cleavage site can improve the extracellular production of cutinase. We also found that extracellular expression of cutinase with pelB signal peptide depends on more than the type II secretion pathway. The phospholipid hydrolysis activity of pelB-cutinase played an important role in extracellular production. Besides, the cutinase was extracellularly expressed by the mediation of HlyAs signal peptide via the Type I secretion pathway. HlyB and HlyD are strain-specific translocation components of the alpha-hemolysin secretion system, which were coexpressed to facilitate the enzyme expression. The cutinase activity from this engineered cell was 2.5 times than that from the type II secretion pathway.

This study investigated that T. fusca cutinase without a signal peptide (cutinaseNS) was expressed in E. coli, indicating the majority of the cutinase activity was located in the culture medium. Biochemical characterization of cutinaseNS has similar to those of the wild type. The majority of inactive cutinaseNS was located in the cytoplasm of E. coli. Furthermore, T. fusca cutinase was confirmed to have hydrolysis activity toward phospholipids, an important component of the cell membrane. Compared with cells expressing the inactive cutinaseNS, cells expressing cutinaseNS showed increased membrane permeability and irregular morphology, but no obvious cell lysis was observed in this process. Based on these results, a hypothesis was proposed to explain the underlying mechanism for the extracellular release of cutinaseNS as “cell leakage induced by the limited phospholipid hydrolysis of cutinaseNS”. The increased membrane permeability could also enhance the extracellular expression of recombinant secretory enzymes and make the extracellular expression of recombinant cytosolic enzymes realized. This novel strategy will have significant potential application in the large-scale preparation of cutinase and other industrial proteins.

Fermentation Strategy for Cutinase

Yan Huang

Cutinase: Preparation and Application, 27 November 2021, Page 87-99

The fermentation conditions for the expression of cutinase by the mediation of pelB signal peptide have been optimized in this study, such as the concentration of the inducer isopropyl \(\beta\)-D-1-thiogalactopyranoside (IPTG) and the induction temperature, as well as the addition of glycine and surfactant sodium bodeoxycholate (TDOC). The highest activity of cutinase in the culture medium reached 149.2 U/mL. To enhance the extracellular expression of cutinase, the recombinant E. coli expression system was constructed using the codon-optimized cutinase gene. After that, the induction strategy was optimized in a 3-L fermentor. Results showed that the optimal induction condition was at a dry cell weight of 13.0 g/L, and the induction strategy was that IPTG is added once in a final concentration of 25.0 \(\mu\)M, and lactose is fed at a rate of 0.5 g/L·h. In this condition, an extracellular cutinase activity of 2258.5 U/mL (5.1 g/L) was achieved, which was the highest cutinase production ever reported.

Application of Cutinase in Textile Industry

Zhan Zhi Liu

Cutinase: Preparation and Application, 27 November 2021, Page 100-121

T. fusca cutinase treatment has become a promising eco-friendly alternative to the chemical method in the textile industry, which was applied as a pretreatment method in wool fiber followed by protease treatment. Cutinase pretreatment could increase the efficacy of protease treatment, which was able to improve the wool fiber, such as the wettability, dyeability, and shrink-resistance. Meanwhile, mild oxidation and cutinase pretreatment exhibited more cooperative effects on wool processing compared with the individual cutinase pretreatment. In addition, this study showed that two fusion proteins, cutinase-CBMCel6A and cutinase-CBMCenA which were generated through the fusion of cutinase and carbohydrate-binding module (CBM), had potential applications in bioscouring. Cutinase-CBMs that combined with pectinase had a greater effect on cotton fiber than that of cutinase alone. Our result also showed that both cutinase-CBMs could result in the formation of carving characters on the surface of cellulose acetate fibers, whose modification effect was greater than that of cutinase alone. This study might provide a foundation for the potential application of cutinase in the textile industry.

Application of Cutinase in Papermaking Industry

Zheng Fei Yan

Cutinase: Preparation and Application, 27 November 2021, Page 122-137

Recycling of waste paper can solve problems of resource and energy shortages, which was considered as an effective and low-cost method. So far, deinking and adhesive removal have become the key factor in the recovery and utilization of waste paper. Enzymatic treatment is less polluting and eco-friendly, which has drawn extensive attention. This study provides a potential strategy for biodeinking by using cutinase. The brightness of the deinked papers realized 42.01% for 8 U/g T. fusca cutinase at pH 8 and 60\(^{\circ}\)C for 30 min; 41.62% for 8 U/g F. solani pisi cutinase at pH 8.5 and 35\(^{\circ}\)C for 30 min. These brightness values are higher than that for chemical deinking by 5.13 and 4.38%, respectively. During the recycling of waste paper, the accumulation of polyacrylates could decrease the quality of the recycled paper and increase the usage of circulating water by the formation of tacky substances known as stickies. Cutinases have the hydrolytic capacity for polyacrylates, which can minimize or eliminate the deposition of stickies. T. fusca cutinase favored the hydrolysis of PEA over PMA dispersions at 0.5 mg/mL, which could limit the turbidity decrease to about 1.0%. Meanwhile, the optimal dosage of T. fusca cutinase was lower than those of F. solani pisi and Humicola insolens cutinases.

Short-chain esters are commonly used as fruit flavorings in the food industry. T. fusca cutinase was used for the synthesis of aliphatic esters, and the maximum yields of ethyl caproate were 99.2% at the optimal condition: 50 U/mL T. fusca cutinase, 40\(^{\circ}\)C, and 0.5% water content, representing the highest yield of ester synthesis. The esterification that catalyzed by cutinase displayed strong tolerance for water content (up to 8%) and acid concentration (up to 0.8 M), respectively. The yields of ester synthesis remained above 80% at substrate concentrations \(\le\) 0.8 M. Moreover, ester yields were achieved more than 98% and 95% for acids of C3-C8 and alcohols of C1-C6, respectively, indicating extensive chain length selectivity of the cutinase. T. fusca cutinase has the superior ability to catalyze the synthesis of short-chain esters, which provided the basis for the industrial production of short-chain esters.

Polyethylene terephthalate (PET), as a major kind of polyester, has become a large source of plastic pollution with an increase of consumption. Currently, different strategies have been exerted for PET degradation, such as enzymatic degradation. To improve degradation efficiency, molecular modification has been proven to enhance the activities of PET degrading enzymes. This study modified T. fusca cutinase by site-directed mutagenesis to enhance its activity on PET fiber. Mutants I218A and Q132A/T101A exhibited considerably higher degradation activities on PET. Cutinase-CBM mutants could also increase degradation activity on PET by modifying CBM binding sites, which exhibited obvious significant improvement in binding affinity and degradation activity toward PET fiber. In addition, this study also summarized the present situation and progresses of PET plastics degradation by enzymatic treatments. Several strategies have been proposed to enhance the degradation performance of enzymes on PET plastics.