Endo-?-1,3-Glucanase Expression In Pichia Pastoris And Hydrolization Of Curdlan For Oligoglucosides Production

Curdlan is a water-insoluble?-1,3-glucan composed of?-1,3-glucosidic bonds which can be produced with fermentation by Agrobacterium sp.ATCC31749.Our laboratory has been engaged in curdlan fermentation and characteristic studies for more than 30 years.We firstly discovered that endo-?-1,3-glucanase expression by Trichoderma harzianum CECT 2413 can effectively hydrolyze curdlan for producing a series of oligoglucosides.?-1,3-oligoglucosides is composed of?-1,3-glucosidic bonds.As a new class of physiological-actived substances,functional oligosaccharides are both antibacterial and antiviral.It can improve immune activity and other biological functions.Thus,it has great potential applications in many fields such as disease diagnosis and prevention,nutrition and health care,etc.However,?-1,3glucanase produced by Trichoderma harzianum ferementation suffers the problems like low enzyme activity of endo-?-1,3-glucanase,complicated separation and purification procedures,and high production costs.In this study,Pichia pastoris was firstly used to express the endo-?-1,3-glucanase from Trichoderma harzianum.In addition,optimization of culture and expression conditions,enzymatic properties,and oligosaccharide hydrolysis properties were studied.As a result,an efficient enzymatic hydrolysis method for the preparation of?-1,3-oligoglucosides by curdlan was establish,which can effectively reduce the production cost,and is expected to replace the traditional acid hydrolysis method.The main research results include:1.Firstly,the endo-?-1,3-glucanase gene from Trichoderma harzianum was synthesized by whole gene technology and the recombinant yeast expression vector pPIC9K-BGN13.1was constructed.The recombinant plasmid pPIC9K-BGN13.1 was linearized by Nco?and transformed into Pichia pastoris KM71 by electroporation.The glucanase gene was successfully integrated into yeast chromosomal DNA through phenotypic screening,^geneticineneticin resistance screening,PCR,SDS-PAGE identification.The expressed recombination endo-?-1,3-glucanase can hydrolyze curdlan to obtain oligosaccharides with DP?degree of polymerization?4-6.2.Secondly,the flask fermentation conditions of recombinant yeast were studied.The optimum fermentation medium was:1%yeast extract,2%tryptone,0.3%calcium chloride,2%magnesium sulfate and 2%dipotassium hydrogen phosphate,1.34%100 mM YNB and 4×10^-5%biotin.The optimum conditions for induction were:initial pH of induction medium7.2,inoculum volume 2.5%,liquid volume was 500 mL^-11 containing 100 mL fermentation broth,25°C,220 r·min^-1,1.5%addition every 24 h methanol/sorbitol was induced and fermented for 4-5 days.The content of endo-?-1,3-glucanase and the enzyme activity(E_endo)in the fermentation supernatant were 1.26-folds and 1.21-folds higher than the control,respectively.3.In addition,to scale up and optimize the expression of targeted proteins by Pichia pastoris,a low temperature?25°C?and methanol/sorbitol co-feeding strategy was implemented during the methanol induction period in a 7 L bioreactor.The results showed that using this combination induction strategy,the maximum endo-?-1,3-glucanase content and enzyme activity were 1.19-folds and 1.16-folds,respectively,compared with batch using the pure methanol induction strategy at 30°C.Therefore,the expression level and the enzyme activity were enhanced simultaneously with the combined induction strategy.4.Finally,the enzymatic characteristics of the purified enzyme from the fermentation supernatant were studied.The results showed that for the hydrolytic process,the optimum pH is 5.5 and the optimum reaction temperature is 50°C.Recombinant endo-?-1,3-glucanase is stable at pH 4.5⁶.5 and temperature 45⁶0°C?relative enzyme activity is above 80%?.Mg²⁺,Cu²⁺and Zn²⁺can exert positive effect on hydrolytic activity.Ca²⁺,Fe²⁺have less effect on enzyme activity of endo-?-1,3-glucanase while Hg²⁺,Ag⁺can obviously inhibits enzyme activity,followed by K⁺and Fe³⁺.According to the results of MALDI-TOF-MS analysis,oligosaccharide DP was mainly 4⁶,DP 7¹0 contained less,glucose and DP 2³ were almost absent.