The Isolation And Identification Of Four Liubao Tea Fungi, Analyses Of Fermentation Features And The Research On Polygalacturonase Gene Pg?

Liupao tea is a kind of famous black tea in Guangxi,one of its important production procedure is"Wodui" in this period there are many microorganisms growing.This research aimed at deriving the fungal resource from Liupao tea,to further be used in the tobacco products and tea.The content and result are listed below(1)The screening and identification of the strains:?Four fungi that have potential uses were isolated from Guangxi Liupao tea.The method of microscopy observation and molecular identification were implemented to identify the species they belong.They were named as Aspergillus tubi,ngensis GYC 501(GYC 501),Aspergillus chevalieri E 2(E 2),Aspergillus chevalieri E 3(E 3),Aspergilltus cristatus E 6(E 6).?The selected fungi were tested for their physiology and biochemistry,the result showed these fungi could utilize different kinds of polysaccharides and have their own characteristics.Initially determined GYC 501 has pectinase and cellulase activities;E 2 has amylase and cellulase activities;E 3 has amylase activity;E 6 has no activity to be shown.(2)The physiological characteristics of the strains:(1)The growth condition in solid orange peel powder medium and solid tobacco powder medium of three Eurotium species fungi were observed,the result showed they adapted better in solid tobacco powder medium.E 2 couldn't grow in solid orange peel powder medium,E 3 had the fastest growth speed in both medium.?The growth condition in liquid tobacco stem powder medium and liquid tea powder medium of three Eurotium species fungi were observed,they performed better in the medium which has tobacco stem as the nutrient substance.Equally,E 3 had the fastest growth speed in both medium.?The thin-layer chromatography method was used for the liquid fermentation supernatants for three Eurotium strains,the result showed these fungi have hardly excreted metabolites that could be extracted and observed,while when cultured in the tea powder medium they all could excrete three kinds of pigments that emanate pink glow under the ultraviolet.?The enzyme activities of pectinase,cellulase and amylase activities were assayed for the liquid fermentation supernatants from three fungi,the result showed these fungi have low polysaccharide hydrolases activity.On the whole,E 6 had the best activities,while E 3 in the middle and E 2 tailed the queue.?The reducing sugar content,total sugar content,total antioxidant activities,DPPH·eliminating activity and tea polyphenols content were assayed for the liquid fermentation supernatants from three fungi,the results showed:for the liquid tobacco stem powder medium,compared to the control,the total sugar content in the fermented medium dropped significantly,the drop percentage ranged from 16.29-39.98%;the reducing sugar content rose,the increment folds ranged from 2.86-3.89;the amino acids content dropped;the change in the total antioxidant activities were insignificant;the DPPH·scavenging activities rose significantly;the tea polyphenols content rose.For the liquid tea powder medium,compared to the control,the total sugar content in the fermented medium rose significantly,the increment percentage ranged from 2.07-35.25%;the reducing sugar content rose drastically,the increment percentage ranged from 131.70?627.02%;the amino acids content dropped,the decrease percentage ranged from 0.52?32.68%;the total antioxidant activities and the DPPH·scavenging activities rose significantly;the tea polyphenols content rose significantly.(3)The cloning and expression of the genes:?A polygalacturonase gene pg? was cloned from GYC 501,the length of repgll is 1038 bp;it was successfully expressed in Pichia pastoris GS115 with high yield.The rePGII protein consists of 346 amino acids,the 7 day induced enzyme activity reached 5672.85±204.39 U/mL.The actual weight of the protein is 38 kDa,its optimal temperature is 500C,the optimal pH is 5.0,its enzyme activity in the reaction solution with 10 mM Zn2+ is 2.78-times of the control.rePGII is relatively thermal-unstable:if the remaining enzyme activity more than 70%within 30 min is considered stable,rePGII is stable in water solution under 300C,and in pH 5.0 citric acid/sodium citrate buffer containing 20%glycerol under 40?,and in pH 5.0 citric acid/sodium citrate buffer under 20?.The Vmax is 1434±159.20 U/mg,Km is 4.319±0.86 mg/mL.?A ?-glucosidase gene bgN was cloned from GYC 501,the length of rebgN is 1761 bp,reBGN has 587 amino acids,it was successfully expressed in Pichia pastoris GS115.?A?-glucosidase gene bg2E was cloned from GYC 501 but it has no expression in Pichia pastoris GS115.?An endoglucanase gene egB was cloned from GYC 501 but it has no expression in Pichia pastoris GS115.?A pectin methylesterasegene pmeA was cloned from GYC 501 and some amount of polygalacturonase activity was shown in Pichia pastoris GS115.