Enzymatic Characterization And Molecular Improvement Of Fungal Pectinases

Pectic substances are one of the main heteroglycan components of plant cell wall, and arecomprised of linear chains of α-1,4-linked d-galacturonic acid residues with different degrees ofesterification. Pectinases, i.e. pectin methylesterase, polygalacturonase and pectic lyase, are enzymesthat catalyze the hydrolysis of pectic substances. Pectinases are widely used in the food industry toextract and clarify the juice and firm the fruits and vegetables. In feed industry, pectinases aresupplemented as additives to alleviate the anti-nutritional role of pectin. Pectinases are also used intextile, paper making etc. To enrich the gene resources and meet the industrial requirements, this studyaims to obtain novel pectinase genes from fungi of different niches and to improve the enzymeproperties of a polygalacturonase with high specific activity by site-directed mutagenesis.One pectin methylesterase gene (pe8F46) was cloned from Penicillium chrysogenum F46, a fungalstrain isolated from food processing wastewater, and was successfully expressed in Pichia pastoris.Purified recombinant PE8F46showed the optimal activty at pH5.0and40oC. This enzyme is alow-temperature-active enzyme, remaining52%activity even at10oC. In the firming experiment ofpineapple dices, the greatest efficiency,47.6%, was achieved when incubated the fruit with0.75%(w/v)PE8F46and0.4%calcium lactate (w/v) for20min, which was13.7%higher than that of thecommercial counterpart.Two endo-polygalacturonase genes (AM-pg I and AM-pg II) and one exo-polygalacturonase gene(AM-pg III) were cloned from Neosartorya fischeri P1, a fungal strain isolated from acid mine. All thegene products were successfully expressed in P. pastoris. Purified recombinant AM-PG I showed theoptimal activty at pH5.0and50oC. The specific activity of AM-PG I was40,123U/mg, higher than allreported recombinant counterparts. Purified recombinant AM-PG II showed the optimal activty at pH4.0and65oC， higher than other endopolygalacturonases. The enzyme had good thermostability,retaining10%activity after incubation at70oC for10min. Purified recombinant AM-PG III was atypical acid enzyme that showed high activity under acidic conditions. The optimal temperature and pHof AM-PG III were60oC and pH3.5. These N. fischeri P1polygalacturonases having differentcharacters may represent good candidates for potential application in various industries.Based on sequence alignment and homology modeling, some key residues were identified in theendo-polygalacturonase PG I of Achaetomium sp. Xz8with specific activity of28,122U/mg. A total of18mutants related to specific activity, functional pH, and temperature were constructed by site-directedmutagenesis. Mutants T97K and T97R showed increased specific activities of12.01%and42.03%,respectively. The decreased pH optima (5.0) of mutants T97G, T97Q and N136D made themprospective for application in the fruit and vegetable processing industry. Mutations at residues far fromactive site, K31D, K121S, R224T, K294D and their combination, also shifted the pH profile to acid.The optimal temperature of T97Q andT97S were increased by10C, and their thermostability wereimproved as well. These improvements of different enzyme property make PG I more suitable for industrialization.In summary, this study identified four pectinases with application potentials in two fungal strains bygene cloning and achieved heterologous expression, and improved the enzyme properties of PG I bysite-directed mutagenesis. The results not only enrich the gene resources of pectinases, but also provideexcellent materials for industrial applications and basic research.