Study On Fermentation Conditions Of Endo-β-1,3-glucanase Production By Trichoderma Harzianum

β-1,3-glucanase is a kind of hydrolase, including exo-β-1,3-and endo-β-1,3-two types.Their interaction can efficiently and specifically degrade beta-1,3-glucan, therefore widelyused in beer, feed and agricultural fields. Endo-β-1,3-glucanase can cut off β-1,3-glycosidicbond in a random way and degrade polysaccharide into low molecular weight functionaloligosaccharides, which has more application value than exo-β-1,3-glucanase.β-1,3-oligosaccharide is mainly beta-1,3-glycosidic connected by glucose monomers. For itsphysiological function, β-1,3-oligosaccharide can prevent dental caries, enhance immunity,anti-tumor and induce plant resistance to pathogen and so on, thus has attracted more andmore attentions in food, health care products, medicine and agriculture areas.Curdlan is a kind of linear water insoluble β-1,3-glucan with controllability ofproduction and quality, low price and high purity. At present curdlan has become a potentialideal raw materials for large-scale preparation of functional β-1,3-oligosaccharides. It hasreported that endo-β-1,3-glucanase from Trichoderma harzianum fermentation has highhydrolytic activity and can be used to hydrolysis curdlan preparing correspondingoligosaccharides. For its inductivity, the endo-β-1,3-glucanase production quantity of T.harzianum itself is quite low. In addition, as the cell walls of fungi, β-1,3-glucanase can causecell autolysis. How to ensure a large biomass and improve the production ofendo-β-1,3-glucanase and its proportion in β-1,3-glucanase is the focus of this research. Forthis purpose, with T. harzianum GIM3.442as the starting strain, the shaking flaskfermentation conditions optimization, fermentation process control and carbon feedingstrategies were investigated to improve the endo-β-1,3-glucanase production and itsproportion in β-1,3-glucanase, providing theoretical foundation for large-scale enzymaticpreparation of oligosaccharides. Specific contents and results are as follows:1. T. harzianum shaking flask fermentation conditions and the orthogonal experiment L9(34) of carbon source, nitrogen source and phosphate were studied, the results were as follows:pachymaran60g·L-1, tryptone20.0g·L-1, NaNO135.0g·L-1, KH2PO410.0g·L-, MgSO4·7H2O2.0g·L-1, CaCl20.4g·L-1, Mandels trace element solution1mL; The strain was culturedat initial pH6.0, inoculum size of8%, with fluid volume100mL in500mL erlenmeyer flask,28oC,110r·min-1, culture6d. After fermentation, the protein content、the total enzymeactivity of β-1,3-glucanase Etotaland endo-β-1,3-glucanase activity Eendowere4.1times,8.7times and23.0times as compared with initial culture conditions respectively; Eendo/Etotalimproved from0.24to0.63.2. The influences of pH and agitation speed in the process of batch fermentation on T.harzianum producing endo-β-1,3-glucanase were studied. A two stages of pH and agitationspeed control strategy was proposed: in the early stage of the fermentation (0-36h), the pHand agitation speed were controlled at7.0and100r·min-1; in the second phase offermentation (36h-168h), the pH and agitation speed were swiched to5.0and200r·min-1.After fermentation, Etotaland Eendowere2.5times and4.2times as compared withuncontrolled conditions respectively; Eendo/Etotalreached0.71, which increased by65.1%ascompared with batch fermentation (0.43). 3. The compound of glucose and pachymaran and the carbon source adding mode werestudied. Composite results showed that when the content of glucose was30%, the repressioneffect is smallest. Through analysising the influence of different carbon source adding time onenzyme production, a pulse adding carbon source-batch cultivation strategie was put forward:in the early stage of the fermentation logarithmic phase (24h) and medium phase (48h) batchadded glucose, ensure bacteria grow quickly, then added high concentration pachymaran at72h for batch cultivation. Etotal, Eendoreached721.5U·mL-1and466.2U·mL-1, which increasedby185.0%and204.7%compared with the batch fermentation, respectively.4. The partical properties of endo-β-1,3-glucanase were studied. It functioned optimallyat pH5.5and50oC, and was stable on the range of pH4.5-5.5and45oC-55oC, in which therelative enzyme activity was more than90%. The endo-β-1,3-glucanase activity was inhibitedby Ag+, Hg2+, followed by K+, Al3+and Fe3+; and activitied by Mg2+, Mn2+, Cu2+and Zn2+todifferent excent; Ca2+, Fe2+, Ba2+had little effects on enzyme activity. The optimal hydrolysistime was2.5h, at which the proportion of oligosaccharides in total sugar was highest.