INTRODUCTION
Generalities
This feature of cutinase was not observed in most lipases except Candida antarctica lipase B (Longhi et al., 1996). Some of these processes have already been implemented in industry (Carvalho et al., 1998a), while others are still under evaluation at the research level.
Cutinase producing microorganisms
It has been observed by Sebastian et al., (1987) that bacterial cutinase is stable even at 70°C, whereas fungal cutinases lose their activity at 45°C. Interestingly, despite the high level of homology, they showed different biochemical properties and substrate specificity (Chen et al., 2010).
Production of recombinant protein
Statistical approach for optimization of process parameters for
Immobilized metal affinity chromatography (IMAC) using (His)6 tag is one of the popular affinity purification methods used for the purification of recombinant proteins. Biophysical characterization plays an important role in an overall analytical strategy of enzymes, complementing biological and biochemical methods used for process and product development of macromolecules.
Immobilization strategy to enhance the activity and stability of
These techniques are critical for understanding the structure and behavior of proteins under different stress conditions, and they are essential tools for understanding enzyme-substrate binding behavior and performing characterization studies for product development.
Esterification and transesterification reactions catalyzed by cutinase
Tyr (Bacterial cutinase) or Gly and Ser (Fungal cutinase) in the vicinity of the active site, from the formation of the oxyanion hole. In enzyme kinetics, the rate of reaction is measured and the effects of changing reaction conditions are investigated.
Objectives and scope
In the present study, we also attempted to improve the stability and activity of the T. Chitosan, zeolite and Celite-545 were found to be a suitable immobilization support to improve the activity and stability of the cutinase in aqueous medium.
Organization of the thesis
A disulfide bond is present in the monomer (Cys280-Cys298) in the C-terminal region. The overall secondary structure of Est119 is very similar to that of S. In addition, screening, selection and media optimization were investigated for increased production of recombinant cutinases in E. The results are discussed in the next section.
LITERATURE REVIEW
Cutinase: an overview
Enzymatic hydrolysis of quince is one of the first steps in most infections. There are also reports that some plant pollens produce cutinase-like enzymes (Maiti et al., 1979).
Thermobifida fusca: an overview
Cutinases, lipases and esterases: an overview
In general, an esterase is specific for either the alcohol or the acid part of the substrate, but not for both (Fojan, 2000). A classification scheme proposed by Whitaker (1972) for esterases was based on their specificity for the acidic part of the substrate, such as the carboxylesterases that catalyze the hydrolysis of carboxylic acid esters. It appears that one of the key factors contributing to the substrate specificity is the physical state of the substrate.
On the other hand, esterases are divided into groups such as cutinase group, acetylcholine esterase group, and all of them have an α/β hydrolase fold. The putative spatial movement of the lid associated with catalysis will expose hydrophobic patches that are solvent-inaccessible in the closed lid conformation.
Cutinase sources and production
- Cutinase production in wild type organisms
- Cutinase production in recombinant organisms
Wild-type cutinase production is affected by several factors, including the type and concentration of carbon and nitrogen sources, culture pH and temperature, and dissolved oxygen concentration (Du et al., 2007). Pulse-chase experiments showed that the introduction of two exposed hydrophobic patches in cutinase resulted in increased immunoaffinity with immunoglobulin (Ig) binding protein heavy chain (BiP), which could result in retention of cutinase in the endoplasmic reticulum of the CY028 mutant ( Sagt et al., 1998 ). . Multicopy strains showed a 6- to 12-fold increase in extracellular cutinase production compared to single-copy strains (van Gemeren et al., 1995; van Gemeren et al., 1996).
In another work (Chen et al., 2011), glycine and sodium taurodeoxycholate (TDOC) were used to increase the secretion of recombinant T. Su et al., (2012) also worked towards extracellular secretion of recombinant cutinase Tfu 0883 from T.
Purification of cutinases
Because of the small difference in I50 of OTFP and MBTFP (Shiotsuki et al., 1994) for Monolinia fructicola cutinase, OTFP was chosen to elute the affinity column (Wang et al., 2000). Almeida et al., (1998) have reported that efficient and inexpensive purification of cutinase can be achieved in PEG-starch derivatives ATPS (aqueous two-phase systems). Extraction and back extraction of pure cutinase was established by Carneiro-da-Cunha et al., (1996b) using the anionic surfactant, AOT, in isooctane for F .
The optimal conditions for the direct extraction of the enzyme from media containing cell debris resulted in an extraction yield of 54.4% (Carneiro-da-Cunha et al., 1994a; Carneiro-da-Cunha et al., 1996a). Chen et al., (2007) used a two-stage chromatography process for the purification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides.
Cutinase structure
The enzyme was found to be a monomer by SDS-PAGE and gel filtration of the native enzyme obtained via a Sephadex G-100 column. Two disulfide bridges exist in cutinase: one between Cys31 and Cys109 that helps stabilize global molecular folding and the other between Cys171 and Cys178. As a member of the α/β hydrolases, this enzyme possesses an active site composed of the catalytic triad residues Ser126, Asp181 and His194 (Fig. 2.4 B).
The catalytic site is surrounded by the two hydrophobic surfaces composed of residues 87–93 of helix 3 and residues 186–194 representing the loop between helices 9 and 10 as well as the first 3 residues of the latter. Such a model implies that the 196–205 loop must undergo a significant conformational rearrangement to form the active state of the enzyme.
Biochemical properties of cutinase
- Molecular properties
- Substrate specificity
- Effect of inhibitors and metal ions
- Organic solvent, surfactant tolerance
- Effect of pH and temperature on cutinase function
In the two-phase system, triolein conversion was monitored using a monolayer technique (Flipsen et al., 1996). Chen et al., (2010) studied the effect of different metal ions on recombinant cutinase activity from T. Chen et al., (2010) also performed inhibition kinetics for SDS to evaluate its inhibitory efficiency on the three cutinases.
The highest activity for A.brassicicola cutinase was observed at 40°C (Koschorreck et al., 2010) and increasing the temperature above 50°C resulted in complete inactivation of the enzyme. Hunsen et al., (2008) studied the ring-opening polymerizations (ROP) of ε-caprolactone (CL) and ω-pentadecalactone (PDL) using H .
Biophysical properties of cutinase
A study using fluorescence spectroscopy, fluorescence decay curves and fluorescence anisotropy decay of the single Trp of cutinase at different pH values (4 to 8) and temperatures, below and above the melting temperature of the protein, showed that the Trp fluorescence spectrum is red shifted and sharp. increase in intensity (fourfold) upon melting proteins with higher intensity at pH 8, indicating that cutinase is more stable at higher pH. Above the melting temperature, the shortest-lived component disappears, possibly due to the breaking of the Ala-Trp hydrogen bond that holds the Trp near the disulfide bridge. Irradiation with UV light at room temperature breaks the disulfide bridge, while the short-lived component of Trp decay persists, suggesting that irradiation maintains the Ala-Trp hydrogen bond.
Thus, the increase in Trp fluorescence quantum yield is mainly due to the suppression of static quenching by Trp-cystine complexes. Structural analysis of the protein by combining NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent showed that the unusual fluorescence behavior of Trp69 in cutinase is caused by the disulfide bond between Cys31 and Cys109 breaking after irradiation, while the amide -bond the aromatic hydrogen bond between Ala32 and Trp69 remains intact.
Strategies for improvement of cutinase activity and stability
- Genetic engineering strategy
- Immobilization strategy
Recently, Ribitsch et al., (2013) investigated the effect of fusing a cellobiohydrolase I binding module from Hypocrea jecorina (CBM) and polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) with cutinase from T. A BSTR (batch stirred tank reactor) was developed by Gonclaves et al., (1998) for the use of the immobilized cutinase in the hydrolysis of triglycerides in non-conventional media. High conversions were observed using 4% water and 96% organic phase in the medium (Gonclaves et al., 1998).
For the transesterification of butyl acetate with hexanol, the optimal wo range is 5-8 (Carvalho et al., 1997a). Regarding the hydrolysis of triolein, the activity still increased up to wo 30 (Melo et al., 1995b).
Cutinase function and applications
- Cutinase in polymer degradation
- Cutinase in textile related industry
- Cutinase in ester synthesis
- Cutinase in transesterification reactions
- Cutinase in detergent and laundry industry
- Cutinase in biodegradation/detoxification
- Cutinase in dairy industry
- Cutinase in fruit industry
Most chemicals and reagents used in biochemical and biophysical characterization studies (p-nitrophenyl butyrate, p-nitrophenyl palmitate, p-nitrophenyl caproate, p-nitrophenyl valerate, p-nitrophenyl laurate, guanidine hydrochloride, urea, DTT, EDTA, Triton X-100 and PMSF), esterification and transesterification reactions were purchased from Sigma-Aldrich (India). The activity of chitosan-coupled and uncoupled cutinase was measured by the pNPB assay as mentioned in section 3.4.1.1. MNP-bound and unbound cutinase activity was measured by the pNPB assay as mentioned in .
The next section discusses the optimization of the physiochemical parameters for the production of recombinant cutinases in E. The production yield of the recombinant cutinase in the medium containing glucose showed the highest activity 6 hours after induction.
MATERIALS AND METHODS
Chemicals and reagents
All restriction enzymes, polymerases, ligases and dNTPs used in the cloning work were purchased from NEB (USA). All chemicals used in the expression study and enzyme purification experiments were purchased from Sigma-Aldrich (India). Chemicals and reagents used in the secondary development study were of analytical grade and obtained from HiMedia (India) or Merck (India).
All other chemicals used in protein analysis were of analytical grade and obtained from Merck (India).
Microorganisms and vector
Cultivation medium and culture conditions
- Cutinase production from wild type T. fusca
- Cutinase production from recombinant E. coli BL21 (DE3) 76
- Assay for cutinase
- The pNP-ester assay
- The pNMSH assay
- Cutin hydrolysis
- Protein determination
- Dry cell weight (DCW)
- Fluorescence spectroscopy
- Circular dichroism (CD) spectroscopy
- GC analysis of esters
Cutinase activity against p-nitrophenyl esters (pNP ester) was determined by measuring the amount of p-nitrophenol released by hydrolysis of pNP ester. One unit of enzyme activity is defined as release of 1 µmol of p-nitrophenol (pNP) per minute. One unit of enzyme activity is defined as release of 1 µmol of p-nitrophenol (p-NP) per minute.
The method for the preparation of pNMSH and p-nitrophenol standard curve was described in Appendix A.4 and Appendix A.1, respectively. The method of preparation of tomato quince and derivatization of methyl ester was described in Appendix A.5 and Appendix A.6, respectively.
Cloning and expression of cutinase encoding genes from T. fusca
- Choice of host and vector
- Primer design
- Isolation of genomic DNA
- Isolation of plasmid DNA
- PCR amplification
- Restriction digestions
- Ligation reaction
- Preparation of competent cells of E. coli DH5α and E. coli
- Transformation in to E. coli DH5α and E. coli BL21 (DE3) 86
- Expression of cutinase encoding genes in E. coli BL21
Based on the literature, the average amount of solvents commonly used in the reaction medium (up to 40%) was chosen to study the stability of the enzyme. To study the effect of urea on the unfolding of the enzymes, the urea solution was prepared at an amount of 0–8 M in 50 mM KPO4 buffer (pH 8) and enzyme was added to it. Appendix A.9 details the official URL of the various web servers used in the structural analysis.
The buffer was removed and the residual activity of the sample was measured using pNPB assay. The relative activity was expressed as the percentage ratio of the activity of the enzyme without freeze-drying. Relative activity was expressed as the percentage ratio of the enzyme's activity to the first recycling cycle.
4.14 2-D electrophoresis gel showing the pI of purified Cut1 (A) and Cut2 (B). 4.8.2 Effect of pH and temperature on the activity and stability of cutinases.
