论文总字数：20048字

摘 要

本文研究了乙酸盐对D-乳酸发酵的代谢调控。考察了不同浓度盐酸盐对D-乳酸和乙酸生成量的影响，发现2g/L的乙酸钠对D-乳酸产量有最大的促进作用，但随着乙酸盐浓度的增大，虽然乙酸得率进一步降低，但是D-乳酸产量也开始下降，这是因为高浓度的乙酸盐（达到5g/L以上时）会为细胞所利用，而细胞对乙酸盐的利用导致了细胞浓度的急剧下降，而对产酸造成了影响。本文研究发现乙酸盐会降低细胞内的ATP含量，解除ATP对糖酵解途径中关键酶的变构抑制作用而提高糖耗速率。2g/L的乙酸钠的添加导致ATP含量由10.1 umol/gDW降低到了7.43 umol/gDW，ATP含量下降了约26%。考察了乙酸盐对产酸期糖酵解途径中的三个关键酶：己糖激酶HK、磷酸果糖激酶PFK以及丙酮酸激酶PK的活力的影响，发现2g/L的乙酸钠加入，对丙酮酸激酶的影响不大，HK的胞外检测活力从158 U/mg protein 提高到了294 U/mg protein，PFK的活力从168 U/mg protein提高到 322 U/mg protein，分别提高了86%、92%。考察不同浓度乙酸盐对菌体生长的影响，发现加入2g/L的乙酸钠，菌体不但没有出现生长延滞，甚至还出现了微量的上升，这点与以往作用于乙酸生成途径的调控策略不同，没有了生长延滞的缺陷。

关键词：D-乳酸，Sporolactobacillus，乙酸盐，糖酵解通量，ATP水平

The metabolic regulation of acetates to D-lactic acid ferment

Abstract

This article was about the metabolic regulation of acetates to D-lactic acid fermentation. It researched on the effect of production of D-lactic acid and acetic acid under the different concentration of acetates. It was found that 2g/L sodium acetate had the maximum promoting effect on the production of D-lactic acid. As te increasing of the concentration of acetates, the production of acetic acid decreased. However, the production of D-lactic acid will also start to decrease as high concentration of acetates will be utilized for the cells if it reached over 5g/L. The acetates utilize for the cell led a sharp decline in the cell concentration which had effect on production of acid. This research found that acetates will reduce the ATP content in cells and release inhibition of allosteric of key enzyme in the glycolytic pathway which will enhance the rate of sugar consumption. ATP content will drop from 10.1umol/gDW to 7.43umol/gDW(decreased about 26%) as the in addition of 2g/L sodium acetate. This article was focused on effect of activity of three key enzymes(hexokinase HK, phosphofructokinase PFK and pyruvate kinase PK) on the acid production in the glycolytic pathway. The result was that 2g/L sodium acetate had no effect on pyruvate kinase, but the activity of HK was increased from158U/mg protein to 294U/mg protein(about 86% increasing) and the activity of PFK increased from168U/mg protein to 322U/mg protein(approximately 92% increasing). This article also investigated different concentration of acetates effect on bacterial growth. It was found out that there was no delay on bacterial growth after adding 2g/L sodium acetate, and even appeared a small rise trade. This result was different with the previous regulation of acetic acid production in the glycolytic pathway because there was no growth delay defects.

Key Words: D-lactic acid, Sporolactobacillu, Acetate, Glycolytic flux, ATP content

目 录

摘要 ……………………………………………………………………………I

ABSTRACT ……………………………………………………………………II

第一章 文献综述 ……………………………………………………………1

1.1 D-乳酸的理化性质…………………………………………………………1

1.2 D-乳酸的应用………………………………………………………………1

1.2.1 D-乳酸在农业中的应用……………………………………………1

1.2.2 D-乳酸在化学工业中的应用………………………………………2

1.2.3 D-乳酸在聚乳酸材料中的应用……………………………………2

1.3 D-乳酸的生产方法…………………………………………………………2

1.3.1 化学合成法…………………………………………………………3

1.3.2 酶法…………………………………………………………………3

1.3.3 微生物发酵法………………………………………………………3

1.4 立题目的和意义……………………………………………………………4

1. 材料与方法 ……………………………………………………………6

2.1 主要试剂与仪器……………………………………………………………6

2.1.1 主要试剂……………………………………………………………6

2.1.2 主要仪器设备………………………………………………………6

2.2 实验方法……………………………………………………………………7

2.2.1 菌种活化及扩大培养………………………………………………7

2.2.2 种子培养……………………………………………………………7

2.2.3 发酵…………………………………………………………………7

2.2.4 取样检测……………………………………………………………8

2.2.5 酶活检测……………………………………………………………8

2.3 分析方法……………………………………………………………………8

2.3.1 Agilent高效液相色谱（HPLC）……………………………………8

2.3.2 生物传感器测定葡萄糖，L-乳酸等 ……………………………9

2.3.3 Promega生物发光检测ATP含量 …………………………………9

1. 乙酸盐对D-乳酸发酵的代谢调控………………………………11

3.1 不同浓度的乙酸钠对D-乳酸产量和乙酸生成量的影响 …………… 11

3.2 乙酸盐的加入扰动了胞内能量代谢 …………………………………… 12

3.3 乙酸盐的加入提高了糖酵解通量…………………………………………14

3.4 乙酸盐的加入不会导致常见的生长延滞…………………………………16

第四章 结论与展望 …………………………………………………………19

4.1 结论 ………………………………………………………………………19

4.2 展望 ………………………………………………………………………19

参考文献 …………………………………………………………………………20

致谢 ………………………………………………………………………………21

1. 文献综述

乳酸世界公认的三大有机酸之一，可分为D型和L型乳酸。其中D-乳酸是重要的手性中间体与有机合成原料, 是多种手性物质的前体, 广泛应用于制药、化妆品和高效低毒农药及除草剂等领域的手性合成^[1]， 市场前景广阔。

请支付后下载全文，论文总字数：20048字