土壤杆菌-毕赤酵母耦合培养直接生产热凝胶低聚糖  

Direct production of curdlan oligosaccharides by coupled fermentation system of Agrobacterium sp.-Pichiapastoris

作　　者：李菲菲 金树霞 朱莉 詹晓北[1] 赵玥[1] 刘丽萍 高敏杰[1] Feifei LI;Shuxia JIN;Li ZHU;Xiaobei ZHAN;Yue ZHAO;Liping LIU;Minjie GAO(Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China;Wuxi Glyco Biotechology Co., Ltd., Wuxi, Jiangsu 214125, China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]无锡格莱克斯生物科技有限公司,江苏无锡214125

出　　处：《过程工程学报》2019年第4期801-808,共8页The Chinese Journal of Process Engineering

基　　金：国家自然科学基金资助项目(编号:31301408);江南大学自主科研计划重点项目(编号:JUSRP51632A);江苏省政策引导类计划(产学研合作)前瞻性联合研究项目(编号:BY2016022-15)

摘　　要：为提高热凝胶低聚糖生产效率,构建了土壤杆菌-毕赤酵母耦合培养体系,其中土壤杆菌代谢产物热凝胶可被毕赤酵母分泌的内切-β-1,3-葡聚糖酶利用直接生产热凝胶低聚糖。用基于不同启动子(AOX1,GAP,FLD)调控的毕赤酵母重组菌株分泌表达内切-β-1,3-葡聚糖酶BGN13.1a,验证其均能有效水解热凝胶得到热凝胶低聚糖。在此基础上,选取GAP启动子调控的毕赤酵母工程菌与土壤杆菌耦合培养,通过设计两种物种之间的共生关系实现稳定共培养并生产聚合度为17~22的热凝胶低聚糖,产量为4.278 g/L。Curdlan oligosaccharides are widely used in biomedicine and food.Agrobacterium sp.ATCC 31749 can produce curdlan under nitrogen-deficient conditions,and endo-β-1,3-glucanasecan hydrolyzecurdlan to curdlanoligosaccharides.To improve the production efficiency of curdlanoligosaccharides,a coupled fermentation system of Agrobacterium sp.-Pichiapastorisis constructed,in which the Agrobacterium sp.metabolite curdlan can be directly used to produce curdlan oligosaccharides by endo-β-1,3-glucanase(secreted by Pichiapastoris).This microbial consortium omits the steps of extraction,purification,and drying of curdlan and endo-β-1,3-glucanase,and it has considerable potential for use in the industrial production of curdlan oligosaccharides.The commercially available β-1,3-glucanase is a complex enzyme(it contains a variety of exoglucanases and endoglucanases),and the specific endoglucanase is difficult to obtain.In is work,to avoid the effects of exo-β-1,3-glucanase,the endo-β-1,3-glucanase(BGN13.1)was expressed in PichiapastorisGS115 by different promoters(AOX1,GAP and FLD),and they were all verified to be effective in hydrolyzing curdlan to curdlan oligosaccharides;the endo-β-1,3-glucanase enzyme activities reached51.24,49.64,and 46.99 U/mL,respectively.On this basis,the Pichiapastorisengineered by the GAP promoter was selected for co-culture with Agrobacterium sp.to avoid the methanol-induced process,and soybean sprout extract was the optimal source of nitrogen for both curdlan and endo-β-1,3-glucanase production.In the end,the coupled fermentation system was divided into two stages to produce the best culture effect:Agrobacterium sp.cell growth stage(pH=7.0),and curdlan oligosaccharides production stage(pH=5.6).Potassium phosphate buffer was added to the medium to keep the pH of the fermentation broth within a constant range.When the initial inoculation ratio of Agrobacterium sp.to Pichiapastoriswas 1:2,and fermentation time in shaker flaskswas 79 h,respectively,the maximum production of curdlan oligosaccharides(

关 键 词：热凝胶低聚糖 耦合培养 内切-β-1 3-葡聚糖酶 土壤杆菌 毕赤酵母 

分 类 号：Q819[生物学—生物工程]