谷氨酸棒状杆菌全细胞催化合成Levan果聚糖条件优化分析

doi:10.16431/j.cnki.1671-7236.2020.11.008

摘要/Abstract

摘要： 试验旨在探究微生物合成Levan果聚糖的新途径并优化其合成条件。利用转入果聚糖蔗糖转移酶基因的重组谷氨酸棒状杆菌进行全细胞催化合成Levan果聚糖，并采用响应面法以Levan果聚糖产量为响应值优化催化合成条件。首先设计Plackett-Burman试验从7个相关因素（蔗糖浓度、菌体浓度、转化时间、转化pH、PBS中Ca²⁺和Mg²⁺浓度、Levan果聚糖提取时的乙醇终浓度）中筛选出影响Levan果聚糖产量的3个主要因素（蔗糖浓度、乙醇终浓度和Ca²⁺浓度）；再用筛选出的3个最重要因素进行最陡爬坡试验逼近最大响应值区域；最后运用BBD响应面法优化确定3个主要因素之间的交互作用和最佳合成条件。结果显示，初始试验中Levan果聚糖平均产量为0.069 g/mL。经过响应面试验优化对利用重组谷氨酸棒状杆菌进行全细胞催化合成Levan果聚糖产量影响最大的因素为蔗糖浓度，其次为提取时的乙醇终浓度，最后是转化液中的Ca²⁺浓度。当蔗糖浓度为52.71%、乙醇终浓度为85.31%、Ca²⁺浓度为0.04 g/L时，利用重组谷氨酸棒状杆菌进行全细胞催化合成的Levan果聚糖产量平均值为0.238 g/mL，与模型预测最大值0.233 g/mL相近，转化率约为44%，转化率较初始试验提高了约1.9倍，产量亦较初始试验提高了约3.4倍。研究结果为Levan果聚糖工业化生产提供了依据，并为其在动物医学和营养方面的研究奠定基础。

关键词: 谷氨酸棒状杆菌; 果聚糖蔗糖转移酶; Levan果聚糖; 全细胞催化; 响应面优化

Abstract: This experiment was aimed to explore a new way for microbial synthesis of Levan and optimize its synthesis conditions.Levan synthesis by whole-cell catalysis using recombinant Corynebacterium glutamicum that transferred into levansucrase,and the response surface method were used to optimize the synthesis conditions with Levan yield as the response value.Firstly,the Plackett-Burman design was used to screen out three main factors affecting yield (sucrose concentration,final concentration of ethanol in Levan extraction and Ca²⁺ concentration) from 7 related factors (sucrose concentration,bacterial concentration,transformation time,transformation pH,Ca²⁺ and Mg²⁺ concentration in PBS,and final concentration of ethanol in Levan fructan extraction).Then the three most important factors selected were used for the steepest climbing test to approach the region of maximum response value,and finally use the BBD response surface method to determine the interaction between the three main factors and the best synthesis conditions.The results showed that the average yield of Levan was 0.069 g/mL in the initial experiment.The response surface method was used to optimize the whole-cell biocatalysis of Levan by Corynebacterium glutamate.The most influential factor was sucrose concentration,the second was the final concentration of ethanol,and the last was the Ca²⁺ concentration in the transformation solution.When the sucrose concentration was 52.71%,the final ethanol concentration was 85.31%,and the Ca²⁺ concentration was 0.04 g/L,the average yield of Levan synthesized by recombination Corynebacterium glutamate was 0.238 g/mL,which was close to the model's predicted maximum value of 0.233 g/mL,and the conversion rate was about 44%.The conversion rate was 1.9 times higher than the initial test,and the yield was 3.4 times higher than the initial test.The results provided the basis for Levan industrial production,and laid the foundation for its research in animal medicine and nutrition.

Key words: Corynebacterium glutamicum; levansucrase; Levan; whole-cell biocatalysis; response surface methodology

中图分类号:

S852.23

吴昕雨, 严琳, 伍红. 谷氨酸棒状杆菌全细胞催化合成Levan果聚糖条件优化分析[J]. 中国畜牧兽医, 2020, 47(11): 3474-3483.

WU Xinyu, YAN Lin, WU Hong. Optimizing Conditions Analysis of Levan by Whole-cell Biocatalysis with Corynebacterium glutamicum[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3474-3483.

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