重组木聚糖酶xynP N-末端对酸稳定性的影响及在苹果汁澄清中的应用  

Effects of Recombinant Xylanase xynP N-terminal on Acid Stability and Application in Clarification of Apple Juice

作　　者：覃池 刘书亮[1] 李建龙[1] 胡凯弟 赵宁 李琴 Qin Chi;Liu Shuliang;Li Jianlong;Hu Kaidi;Zhao Ning;Li Qin(College of Food Science,Sichuan Agricultural University,Ya'an 625000,Sichuan)

机构地区：[1]四川农业大学食品学院,四川雅安625000

出　　处：《中国食品学报》2024年第11期163-175,共13页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31901634);四川省科技厅项目(2022NSFSC1739);省级大学生创新创业训练计划项目(S202310626099)。

摘　　要：为探究N-末端对木聚糖酶酸稳定性的影响,从NCBI数据库中筛选1段基因序列进行克隆表达,设计响应面试验获得最佳表达条件。从N-末端入手,采用重叠延伸PCR技术对N-末端置换构建突变体,表征木聚糖酶活力及相关酶学性质。将该酶应用于苹果汁中,研究其澄清效果。结果表明:在100 mL TB培养基中接入1.5%种子液,诱导时机为3.5 h,添加200μL 500 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG),在20℃诱导21.6 h得到最佳木聚糖酶xynP表达条件,其酶活为1884 U/mL。木聚糖酶xynP和突变酶xynP-1的最适温度均为40℃,最适pH值分别为4.5和5.0。在不同pH值处理后,突变酶xynP-1在较酸的条件下(pH=4.0)稳定性略弱于野生型木聚糖酶xynP,而随着pH值的增加(pH 4.5~5.5)其酸稳定性均高原酶xynP,说明N-末端对木聚糖酶的酸稳定性有一定影响。金属离子Pb2+和化学试剂十二烷基硫酸钠(SDS)、乙二胺四乙酸(EDTA)对2种酶酶活力有明显的抑制作用。以榉木木聚糖为底物时测得两种酶的K_(m)值分别为分别为1.72 mg/mL和2.57 mg/mL;v_(max)值分别为0.93 nmol/(mg·min)和1.15 nmol/(mg·min),突变酶的底物亲和力和催化效率均不及原酶。将2种酶应用于苹果汁中,加原酶xynP和突变酶xynP-1后,果汁透光率分别由24.33%提高到90.03%和95.70%,说明添加木聚糖酶xynP对苹果汁有显著的澄清效果。该研究结果为后续探究木聚糖酶在果汁加工中的应用提供了参考。In order to explore the effects of N-terminal on acid stability of xylanase,this paper cloned and expressed a gene sequence screened from NCBI database,designed response surface test to obtain the best expression conditions,and constructed N-terminal displacement mutants from N-terminal by overlapping extension SORPCR technology,characterized its enzyme activity and related enzymatic properties,and finally applied it to apple juice to explore the clarification effect.The results showed that the optimal xylanase xynP expression condition was 1884 U/mL by adding 1.5%seed solution into 100 mL TB medium,the induction time was 3.5 h,200μL of 500 mmol/L isopropylβ-D-thiogalactoside(IPTG)was added,and the enzyme activity was induced at 20℃for 21.6 h.The optimum temperature of xylanase xynP and mutant xynP-1 was 40℃,and the optimum pH value was 4.5 and 5.0,respectively.After different pH value treatments,it was found that the stability of mutant enzyme xynP-1 was slightly weaker than that of wild type xylanase xynP under acid conditions(pH=4.0),but with the increase of pH(pH 4.5~5.5),its acid stability was all plateau enzyme xynP,indicating that N-terminal had an impact on the acid stability of xylanase.Metal ions Pb2+and chemical reagents sodium dodecyl sulfate(SDS)and ethylene diamine tetraacetic acid(EDTA)have a significant inhibitory effect on the activity of the two enzymes.When beechwood xylan was used as substrate,the K_(m)values of the two enzymes were 1.72 mg/mL and 2.57 mg/mL,respectively.The v_(max) values were 0.93 nmol/(mg·min)and 1.15 nmol/(mg·min),respectively,indicating that the substrate affinity and catalytic efficiency of the mutant enzyme were lower than those of the original enzyme.Two enzymes were applied to apple juice.After the addition of the original enzyme xynP and the mutant enzyme xynP-1,the light transmittance of the juice increased from 24.33%to 90.03%and 95.70%,respectively,indicating that the addition of xylanase xynP had a significant clarifying effect on apple juice.The results

关 键 词：木聚糖酶 N-末端替换 酶学性质 果汁澄清 

分 类 号：TS255.44[轻工技术与工程—农产品加工及贮藏工程]