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Title: Heterologous Expression of Grape Gene Encoding Pectin-Degrading Polygalacturonase; Industrial Relevance
Authors: Nazir, Zahra
Keywords: Biological & Medical Sciences
Issue Date: 2020
Publisher: University of the Punjab , Lahore
Abstract: Pectinolytic enzymes, widespread in plants, fungi and microorganism, gain commercial importance for improving the yield and nutraceutical properties of juice processing industry, degumming of fibre plants and maximum oil recovery. Pectinolytic enzymes degrade the complex polysaccharide polymers of plant cell wall into simpler monomer like D-galacturonic acids. In current study, polygalacturonase (PG), pectin methyl esterase (PME) and pectate lyase (PL) from grape skin were purified by salting out with ammonium sulphate, gel filtration chromatography on Sephadex G-75 column and ion exchange on Q-Sepharose column chromatography. Protein content was determined by Bradford method. Polygalacturonase appeared as a protein of 47 kDa molecular weight by SDS-PAGE. The optimum pH of purified polygalacturonase activity was found to be 4.5 and stable within pH range 3.5-5.5. Temperature dependent studies revealed temperature optimum of enzyme to be 400C and stable up to 600C. Km and Vmax values were calculated for PG which were found out to be 0.128 mg ml-1 and 0.65 U ml-1 . Among substrates, polygalacturonic acid was established as the best substrate for polygalacturonase showing its specificity in the hydrolysis of polysaccharide galacturonate chain. The presence of Na+1 and K+1 enhanced polygalacturonase activity upto 110% and 130%, respectively when used at a concentration of 1mM, however, the enzyme was almost completely inhibited by Pb+2 and Hg+2 . Purified PME enzyme after electro-elution was displayed on SDS-PAGE as a monomeric protein of 30kDa mass. The optimum pH for PME was found out to be 7.5 and PME was stable within pH range 7.0-8.0. Optimum temperature for PME was 30°C and enzyme was stable up to 40°C. Km and Vmax values were calculated for PME which were found out to be 0.142 mg ml-1 and 0.67 U ml-1 . Purified PL enzyme after electro-elution was displayed on SDS-PAGE as a protein of molecular mass 40kDa. The optimum pH for PL was found out to be 8.0 and PL was stable within pH range 7.0-8.5. Optimum temperature for PL was 37°C and PL enzyme was stable up to 50°C. Considering the significance of pectinolytic enzymes in industrial applications, PG was cloned and expressed in E. coli BL21 (DE3) for PG production in bulk to be used in industrial applications. In the present study, total RNA was isolated from grape skin and synthesis of cDNA was performed. Primers were designed by software DNA Star, NEB Cutter and Oligo Analyzer Tool. ix PCR reaction was performed to amplify PG gene from grape skin and cloned into E. coli DH5α using pTZ57R/T which is a cloning vector. Resulting recombinant plasmid was transformed into competent cells of E. coli DH5α. Positive clones were screened by blue white screening and confirmed by colony PCR and restriction analysis. E. coli expression system was used for expression of PG gene. After confirmation, PG gene (1335bp) was expressed in E. coli BL21 (DE3) employing expression vector pET28a(+). IPTG was utilized as an inducer for the induction of recombinant protein expression. Effect of various IPTG concentrations (0.2, 0.5, 1.0 & 1.5 mM) and auto-induction on expression was studied and maximum expression was obtained with 1.0 mM IPTG concentration Molecular weight of 47 kDa was determined by sodium dodеcylsulphate polyacrylmidе gel electrophoresis, DNS method was used to evaluate the recombinant enzyme activity and zymogram was used to determine the activity of the expressed protein by using its substrate polygalacturonic acid. After sequencing of cloned product, sequencing results were analyzed by BioEdit software and nucleotide sequence was translated into aminoacid sequence by Expasy Translate tool. 3D-modelling and docking analysis of sequenced PG protein was performed by Raptor X, Galaxy Refine and CASP10 server. Auto dock Vina and Discovery Studio visualizer were employed to view docking results. Fruit juices have great nutraceutical potencial and immense health benefits are associated with natural products. Important features of natural products can be improved to a great deal by employing new trends of biotechnology. Physico-chemical properties of PG treated apple juice were determined by employing various methods. Expressed polygalacturonase showed pectinolytic effect in juice clarification when used at concentration 5.0 U/ml, incubation time 60 min and incubation temperature 500C. Maximum clarity of apple juice (95% transmittance), juice yield (77%), total soluble solids (0Brix up to 14.8), total phenolic content (13.82 mg/ml) and free radical scavenging activity of apple juice (77%) were improved after clarification. Whereas, viscosity of apple juice after PG treatment was reduced from 2.0 to 1.09 milli pascal second with viscosity reduction (45.5%) and remarkable decrease in turbidity of juice was also observed from 33.9 to 3.9 NTU. Color of juice is an important sensory attribute for consumers, in our study, color of apple juice after PG treatment appeared to be very light which is good for consumer point of view. Antimicrobial activity of PG treated apple juice was also enhanced (up to 90%) due to rise in total phenolic content of apple juice after PG treatment. To understand the outcome of juice clarification parameters, statistical analysis (Response Surface Methodology) was conducted by minitab and box plots based on surface response methodology were developed which demonstrated that independent variables like PG x concentration, temperature and incubation time had significantly influenced the dependent variables (physico-chemical properties) and improved the overall quality of apple juice. Eco-friendly biomaterials have great potential to be used at large scale for the preparation of various commercial items. Fiber obtained from natural sources is proved to be a good replacement for expensive artificial fiber. No health hazards are associated with natural biopolymer as it is biodegradable but with good tensile strength. Banana pseudo stem, a waste product after crop harvest, has got excellent features to become a commercial fiber. In current trial, pectin hydrolysis of banana pseudo stem by PG enzyme was performed to obtain better quality fiber from banana stem. Reducing sugar level after PG treatment was enhanced which showed pectin degradation in banana pseudo stem. To check the effectiveness of PG treatment on banana pseudo stem, morphological studies were performed by scanning electron microscope which clearly displayed pectin degradation within the cell architecture of banana pseudo stem after PG treatment. Hence, plant polygalacturonase expressed in E. coli expression system would be useful as a potential candidate for its biotechnological applications in fruit juice clarification and fiber industry. This study suggests a need for implication this technology in other related fields at commercial scale.
Gov't Doc #: 20722
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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