Please wait a minute...
 
 
食品与发酵工业  2022, Vol. 48 Issue (9): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.029681
  本期目录 | 过刊浏览 | 高级检索 |
结冷胶裂解酶在毕赤酵母中的异源表达及其性质和应用
段飞扬1, 王力2, 詹晓北1, 蒋芸1, 李志涛1, 高敏杰1*
1(江南大学 生物工程学院,江苏 无锡,214122)
2(江苏艾津作物科技集团有限公司,江苏 南京,211511)
Heterologous expression of gellan lyase in Pichia pastoris and its properties and application
DUAN Feiyang1, WANG Li2, ZHAN Xiaobei1, JIANG Yun1, LI Zhitao1, GAO Minjie1*
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
2(Jiangsu Aijin Agrochemical Co.Ltd., Nanjing 211511, China)
下载:  HTML   PDF (2090KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 结冷胶寡糖由结冷胶降解所得,具有益生活性、植物诱导抗病性等多种生物学功能,其制备意义重大。该研究通过构建重组毕赤酵母以实现结冷胶裂解酶异源表达,并应用于裂解结冷胶制备结冷胶寡糖。重组菌株经筛选鉴定,首先于摇瓶水平诱导表达结冷胶裂解酶,获得酶活力最高为266.4 U/L的菌株;随后在7 L发酵罐中进行高密度发酵,酶活力达954.6 U/L。对重组结冷胶裂解酶进行分离纯化并研究其酶学性质,结果表明,酶促降解反应最适催化温度和pH分别为45 ℃和7.5,在45~50 ℃和pH 7.0~9.0下稳定,Zn2+对酶活性略有促进作用,Al3+对酶促反应具有较强的抑制作用。通过薄层色谱法联用基质辅助激光解析电离飞行时间质谱分析降解产物,该结冷胶寡糖聚合度为4。该研究首次在毕赤酵母中成功表达Bacillus sp.GL1来源结冷胶裂解酶,特异性降解结冷胶制备寡糖,具有一定的工业应用潜力和指导意义。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
段飞扬
王力
詹晓北
蒋芸
李志涛
高敏杰
关键词:  结冷胶裂解酶  毕赤酵母  异源表达  酶学性质  结冷胶寡糖    
Abstract: Gellan oligosaccharide can be degraded from gellan gum, and which has many biological functions such as prebiotic activity, plant induced disease resistance and so on. This study focused on the heterologous expression of gellan lyase by constructing recombinant Pichia pastoris and applied to the preparation of gellan oligosaccharide. After screening and identification, the recombinant strain of P. pastoris GS115-pPIC9K-nGL1 was first induced to express the gellan lyase at shake flask level, and the highest enzyme activity was 266.4 U/L; then, high-density fermentation was carried out in a 7 L bioreactor, and the enzyme activity reached 954.6 U/L, which was 3.58 times higher than that of shaking flask. The enzymatic properties were studied after the recombinant gellan lyase was isolated and purified. The analysis results indicated that the optimal temperature and pH of hydrolysis reaction were 45 ℃ and 7.5. The activity of gellan lyase was relatively stable at 45-50 ℃, pH 7.0-9.0, and it was slightly enhanced by Zn2+. In addition, Al3+ had a strong inhibitory effect on enzymatic reaction. The degradation product was analyzed by thin-layer chromatography combining with matrix-assisted laser analysis ionization time-of-flight mass spectrometry, the result showed that the polymerization degree of gellan oligosaccharide was 4. The gellan lyase derived from Bacillus sp. GL1 was heterologous expressed in P. pastoris GS115 for the first time, and the specific degradation of gellan gum to produce gellan oligosaccharide may have certain potential in industrial applications.
Key words:  gellan lyase    Pichia pastoris    heterologous expression    enzymatic property    gellan oligosaccharide
收稿日期:  2021-10-12      修回日期:  2021-11-15           出版日期:  2022-05-15      发布日期:  2022-05-26      期的出版日期:  2022-05-15
基金资助: 国家重点研发计划“绿色生物制造”重点专项(2021YFC2101100)
作者简介:  第一作者:硕士研究生(高敏杰副教授为通信作者,E-mail:jmgao@jiangnan.edu.cn)
引用本文:    
段飞扬,王力,詹晓北,等. 结冷胶裂解酶在毕赤酵母中的异源表达及其性质和应用[J]. 食品与发酵工业, 2022, 48(9): 1-7.
DUAN Feiyang,WANG Li,ZHAN Xiaobei,et al. Heterologous expression of gellan lyase in Pichia pastoris and its properties and application[J]. Food and Fermentation Industries, 2022, 48(9): 1-7.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.029681  或          http://sf1970.cnif.cn/CN/Y2022/V48/I9/1
[1] 李传宝, 黄金, 何军邀, 等.微生物多糖结冷胶的发酵过程优化[J].食品与发酵工业, 2013, 39(4):76-80.LI C B, HUANG J, HE J Y, et al.Optimization of fermentation process of microbial polysaccharide-gellan gum[J].Food and Fermentation Industries, 2013, 39(4):76-80.
[2] 李小勇, 李洪军, 贺志非, 等.结冷胶及其在食品工业中的应用[J].食品与发酵工业, 2005, 31(6):94-96.LI X Y, LI H J, HE Z F, et al.Gellan gum and its application in food industry[J].Food and Fermentation Industries, 2005, 31(6):94-96.
[3] 汤坚, 詹晓北, 朱莉, 等.结冷胶溶液凝胶特性的研究[J].中国食品学报, 1998(1):77-82.TANG J, ZHAN X B, ZHU L, et al.Study on gel property of gellan gum solution[J].Journal of Chinese Institute of Food Science and Technology, 1998(1):77-82.
[4] XU J J, WANG R Y, ZHANG H T, et al.In vitro assessment of prebiotic properties of oligosaccharides derived from four microbial polysaccharides[J].LWT, 2021, 147:111544.
[5] SALACHNA P, MIZIELIN'SKA M, SOBÓL M.Exopolysaccharide gellan gum and derived oligo-gellan enhance growth and antimicrobial activity in Eucomis plants[J].Polymers, 2018, 10(3):242.
[6] SALACHNA P, GRZESZCZUK M, MELLER E, et al.Effects of gellan oligosaccharide and NaCl stress on growth, photosynthetic pigments, mineral composition, antioxidant capacity and antimicrobial activity in red perilla[J].Molecules, 2019, 24(21):3925.
[7] QIN Z, CHEN Q M, LIN S, et al.Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation[J].Food Chemistry, 2018, 253:139-147.
[8] LI A, LUO H Q, HU T T, et al.Screening and enzymatic activity of high-efficiency gellan lyase producing bacteria Pseudoalteromonas hodoensis PE1[J].Bioengineered, 2019, 10(1):240-249.
[9] MIYAKE O, KOBAYASHI E, NANKAI H, et al.Posttranslational processing of polysaccharide lyase:Maturation route for gellan lyase in Bacillus sp.GL1[J].Archives of Biochemistry and Biophysics, 2004, 422(2):211-220.
[10] LUO S, QIN Z, CHEN Q M, et al.High level production of a Bacillus amlyoliquefaciens chitosanase in Pichia pastoris suitable for chitooligosaccharides preparation[J].International Journal of Biological Macromolecules, 2020, 149(2):1 034-1 041.
[11] MOSER S, STROHMEIER G A, LEITNER E, et al.Whole-cell (+)-ambrein production in the yeast Pichia pastoris[J].Metabolic Engineering Communications, 2018, 7:e00077.
[12] YANG Z L, ZHANG Z S.Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris:A review[J].Biotechnology Advances, 2018, 36(1):182-195.
[13] GAO M J, SHI Z P.Process control and optimization for heterologous protein production by methylotrophic Pichia pastoris[J].Chinese Journal of Chemical Engineering, 2013, 21(2):216-226.
[14] GAO M J, YAN J J, ZHAO Y, et al.Expression of a thermostable β-1,3-glucanase from Trichoderma harzianum in Pichia pastoris and use in oligoglucosides hydrolysis[J].Process Biochemistry, 2021, 107:74-82.
[15] FURUSAWA G, AZAMI N A, TEH A H.Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacterium Catenovulum sp.CCB-QB4[J].PeerJ, 2021, 9:e10929.
[16] GAO M J, YANG G S, LI F F, et al.Efficient endo-β-1,3-glucanase expression in Pichia pastoris for co-culture with Agrobacterium sp.for direct curdlan oligosaccharide production[J].International Journal of Biological Macromolecules, 2021, 182:1 611-1 617.
[17] LI J X, ZHANG P, HONG L, et al.Purification and characterization of a novel hydrolase that can specifically degrade the polysaccharide isolated from green seaweed Ulva prolifera[J].Journal of Ocean University of China, 2019, 18(1):185-192.
[18] NIE Y S, HUANG M Z, LU J J, et al.Impacts of high β-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via 13C metabolic flux analysis[J].Journal of Biotechnology, 2014, 187:124-134.
[19] GAO M J, XU Y, YANG G S, et al.One-step production of functional branched oligoglucosides with coupled fermentation of Pichia pastoris GS115 and Sclerotium rolfsii WSH-G01[J].Bioresource Technology, 2021, 335:125286.
[20] 崔连杰, 李科, 李震宇, 等.寡糖提取分离与质谱结构解析研究进展[J].药学学报, 2020, 55(5):843-853.CUI L J, LI K, LI Z Y, et al.Progress in oligosaccharide extraction, separation and structural analysis by mass spectrometry[J].Acta Pharmaceutica Sinica, 2020, 55(5):843-853.
[21] GUO X, SANG J C, CHAI C C, et al.A lytic polysaccharide monooxygenase from Myceliophthora thermophila C1 and its characterization in cleavage of glycosidic chain of cellulose[J].Biochemical Engineering Journal, 2020, 162:107712.
[22] ITOH T, NAKAGAWA E, YODA M, et al.Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp.str.FPU-7[J].Scientific Reports, 2019, 9(1):14870.
[1] 赵书范, 李琪, 聂红梅, 汪钊, 郑建永. 黑曲霉脂肪酶基因的克隆及其在黑曲霉中的同源表达[J]. 食品与发酵工业, 2022, 48(9): 14-19.
[2] 来欢欢, 张凯悦, 田青, 董晋文, 赵微, 崔美林, 张秀红. 清香酒醅来源Lactobacillus plantarum SL32-2自溶酶的酶学性质及其底物特异性初探[J]. 食品与发酵工业, 2022, 48(7): 64-69.
[3] 李阳阳, 刘松, 尹小强, 堵国成. 黑曲霉木聚糖酶在大肠杆菌中的胞外表达和酶学性质研究[J]. 食品与发酵工业, 2022, 48(6): 1-7.
[4] 杨国帅, 许颖, 詹晓北, 蒋芸, 李志涛, 高敏杰. Microbacterium sp.XT11黄原胶内切酶在毕赤酵母中的异源表达、性质及应用[J]. 食品与发酵工业, 2022, 48(6): 8-14.
[5] 陈宁, 延文星, 路福平, 刘夫锋. 磷脂酶D交联聚集体的制备及其酶学性能研究[J]. 食品与发酵工业, 2022, 48(5): 1-7.
[6] 胡金远, 乔士达, 张萌, 许菲. 富脯氨酸胶原蛋白的重组表达及热稳定性研究[J]. 食品与发酵工业, 2022, 48(5): 15-22.
[7] 冯梦茹, 周化岚, 张建国. 毕赤酵母产类人胶原蛋白发酵条件的优化[J]. 食品与发酵工业, 2022, 48(3): 9-14.
[8] 徐佳, 高廷, 杨彦博, 刘永辉, 田艳杰, 崔彩霞, 郭长江, 周晨妍. A152G突变对GH43家族麦氏交替单胞菌木聚糖酶XynZT-2的影响[J]. 食品与发酵工业, 2022, 48(3): 50-55.
[9] 张献, 冯治平, 张耀, 张雪怡, 杨丽娟. 假丝酵母氨基甲酸乙酯水解酶的分子克隆及酶学性质[J]. 食品与发酵工业, 2022, 48(3): 56-62.
[10] 高宇豪, 吴勇杰, 朱亚鑫, 付静, 徐建国, 王松涛, 徐国强, 张晓梅, 史劲松, 许正宏. 产谷胱甘肽毕赤酵母工程菌的构建及能量调控[J]. 食品与发酵工业, 2021, 47(7): 21-26.
[11] 陶大炜, 宁喜斌. 产α-环糊精葡萄糖基转移酶的菌株筛选、鉴定与酶学性质的初步研究[J]. 食品与发酵工业, 2021, 47(6): 145-151.
[12] 彭燕鸿, 苏爱秋, 黄伟文, 蓝素桂, 杨天云, 谭强. 微生物嗜热脂肪酶研究进展[J]. 食品与发酵工业, 2021, 47(6): 289-294.
[13] 杨胜远, 林谦, 刘淑敏, 苏巧云, 黄慧玲. 屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究[J]. 食品与发酵工业, 2021, 47(5): 28-34.
[14] 吕奎, 贾禄强, 戴京京, 丁健. 通用型毕赤酵母高密度培养策略的网络共享技术[J]. 食品与发酵工业, 2021, 47(5): 92-98.
[15] 钱晓芬, 吴涛, 赵理想, 孙杰, 汪钊, 魏春. 基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响[J]. 食品与发酵工业, 2021, 47(4): 1-6.
[1] .YANG Qian et al . Mutation and functional properties of the histidine residues existed in the conserved regions of Rhizopus oryzae α-amylase[J]. Food and Fermentation Industries, 2017, 43(11): 22 -29 .
[2] WU Xiang-yi et al . Effect of yak milk casein hydrolysates on protein carbonyl content and activity of antioxidant enzymes of oxidative damaged HepG2 cells induced by H2O2[J]. Food and Fermentation Industries, 2017, 43(11): 34 -38 .
[3] LEI Lin et al. Effects of Maturity on the Nutrients in Cabbages[J]. Food and Fermentation Industries, 2017, 43(11): 101 .
[4] WANG Jun et al. Screening methodof37 foodadditivesinfruitcansby UPLC-Q/TOF[J]. Food and Fermentation Industries, 2017, 43(11): 212 .
[5] YU Qing-lin et al. Fermentation optimization of recombinant Yarrowia lipolytica for its efficient succinic acid production[J]. Food and Fermentation Industries, 0, (): 1 .
[6] Zheng Dan et al.. The inhibiting effect of flavonoid “astilbin” on pancreatic lipase[J]. Food and Fermentation Industries, 0, (): 1 .
[7] . Effect of Protein on Quality of Chinese Rice Wine #br# [J]. Food and Fermentation Industries, 0, (): 1 .
[8] . [J]. Food and Fermentation Industries, 2002, 28(4): 64 .
[9] . Effects of Nutritional and Environmental Conditions on the Production of L-Isoleucine by Brevibacterium flavum WL-10[J]. Food and Fermentation Industries, 2002, 28(5): 14 .
[10] . [J]. Food and Fermentation Industries, 2004, 30(9): 103 .
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn