草本纤维生物提取关键酶基因克隆与表达研究
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摘要
提取草本纤维是将草本原料中的纤维与非纤维物质分离开来的过程。草本纤维生物提取是利用微生物所分泌的酶降解原料中非纤维素物质,达到使纤维素分离的目的。草本纤维生物提取效果的好坏,关键在于菌种的优劣。自然界中能降解非纤维素类物质的微生物种类极多,但是能直接用于草本纤维提取的优良菌株很少。如何构建优良的草本纤维提取菌种是一个关键问题。本论文在已有的研究基础上,对草本纤维生物提取关键酶的新基因进行了发掘、对甘露聚糖酶基因的缺失表达、构建复合酶表达体系进行了研究,为草本纤维菌种的成功构建奠定基础。
本论文获得的主要结果如下:
用水解圈法对本实验室的一些产果胶酶菌种资源进行了筛选,同时测定这些菌株发酵液果胶酶酶活,结果筛选出分泌果胶酶能力强的欧文氏杆菌CXJZ-166。参考GenBank上已经登录的果胶酶基因序列,设计引物,采用PCR扩增的方法,从欧文氏杆菌CXJZ-166基因组DNA中克隆了果胶酶基因。该基因序列已经提交NCBI数据库,登录号:EU597234。该果胶酶基因序列全长877bp,共编码277个氨基酸。该果胶酶基因所编码蛋白质预测的二级结构表明,其主要的二级结构是β折叠,无规卷曲、α螺旋的含量很低。
采用缺失表达的方法,对甘露聚糖酶基因进行了系统的缺失表达研究,未见他人的相关报道。结果表明,甘露聚糖酶基因3′单侧缺失最大可以删除387bp(对应129aa)不会使表达产物甘露聚糖酶丧失活性,缺失序列占源甘露聚糖酶基因序列长度的34%;甘露聚糖酶基因5′单侧缺失最多可以删除291bp(对应97aa),剩余部分序列表达产物依然具有甘露聚糖酶活性,删除的片段长度占全长的25.6%;甘露聚糖酶基因5′、3′两侧同时缺失,5′端删除234bp(78aa),3′端删除387bp(129aa),表达产物具有甘露聚糖酶活性,这是本实验所找到的最小的且具有甘露聚糖酶活性的序列片段,即为源甘露聚糖酶基因序列长度的45.1%。
采用并联模式尝试构建复合酶表达体系,是一次新的策略,据现有资料来看,尚属首次。首先构建单质酶的复合表达载体,再通过热激转化法,将单质酶载体整合同一表达宿主中。将两种单质酶载体整合到同一宿主,获得了P-M、P-X、M-X表达体系,结果表明,一种单质酶的表达并不影响另外一种酶。将三种单质酶载体整合到同一宿主,获得的P-M-X表达体系同时分泌果胶酶、甘露聚糖酶、木聚糖酶三种酶,与现有生产用的菌株相比,果胶酶酶活相同,甘露聚糖酶酶活提高41.7%、木聚糖酶酶活提高了350%。脱胶实验表明,P -M-X的脱胶效果远远不如现在生产上所用的菌株。
The extracting of herbaceous fiber is a process to make cellulose apart from non-cellulose. The process is completed by enzyme produced by microbiology. The effect of bio-extracting of herbaceous fiber depends on the strain. Though there are various types microbiology that can secreting enzyme to degrade non-cellulose in the nature, a few kind of microbiology can be directly applied to extracting of herbaceous fiber. The key problem is how to improve the characters of the strain. The paper is on the key enzyme for bio-extracting of herbaceous fiber, including three parts: novel gene, lose expression and constructing compound enzyme expression system.
By comparison the clearing halos and the enzyme activity, CXJZ-166 was the strain of pectinase high, which was the most suitable strain for cloning pectinase gene. By PCR, a fragment of DNA were amplified from the the genome DNA of CXJZ-166.The PCR product were linked with pMD 18-T vector, transformed into E.coli JM109. Clear halos were made by positive clones in pectinase selecting plate. The DNA fragment is 877bp,coding 277aa, had been registered in Genbank under the accession number of EU597234.
Lose expression ofβ-mannanase had been tested systematically. The DNA sequence were shorten to 513bp (the full length 1134bp) in 3′end, accounting for 45% of the full length ,which hasβ-mannanase activity. 291bp had been deleted in 5′end, accounting for 25.6%, the fragment had the mannanase activity,. If some fragment ofβ-mannanase gene were deleted in two ends at the same time, 234bp in 5′and 387bp in 3′could be truncated. This was the most short fragment with mannanase activity in the paper, accounting for 45.1%.
Some kinds of enzyme were integrated into the same strain to secret kinds of enzyme simultaneously. Three kinds of two-enzyme strain were acquired in bio-extracting of herbaceous fiber,which were pectinase, mannanase and xylanase. Three kinds strain producing two kinds of enzyme had been acquired, which were Pectinase-Mannanase strain, Pectinase-Xylanase strain and Mannanase-Xylanase strain. P-M-X compound enzyme expression can secret pectinase,mannanase and xylanase. Mannanase and xylanase activity in fermented liquid of P-M-X were increased by 41.7% and 350% than the strain for production now.
本论文获得的主要结果如下:
用水解圈法对本实验室的一些产果胶酶菌种资源进行了筛选,同时测定这些菌株发酵液果胶酶酶活,结果筛选出分泌果胶酶能力强的欧文氏杆菌CXJZ-166。参考GenBank上已经登录的果胶酶基因序列,设计引物,采用PCR扩增的方法,从欧文氏杆菌CXJZ-166基因组DNA中克隆了果胶酶基因。该基因序列已经提交NCBI数据库,登录号:EU597234。该果胶酶基因序列全长877bp,共编码277个氨基酸。该果胶酶基因所编码蛋白质预测的二级结构表明,其主要的二级结构是β折叠,无规卷曲、α螺旋的含量很低。
采用缺失表达的方法,对甘露聚糖酶基因进行了系统的缺失表达研究,未见他人的相关报道。结果表明,甘露聚糖酶基因3′单侧缺失最大可以删除387bp(对应129aa)不会使表达产物甘露聚糖酶丧失活性,缺失序列占源甘露聚糖酶基因序列长度的34%;甘露聚糖酶基因5′单侧缺失最多可以删除291bp(对应97aa),剩余部分序列表达产物依然具有甘露聚糖酶活性,删除的片段长度占全长的25.6%;甘露聚糖酶基因5′、3′两侧同时缺失,5′端删除234bp(78aa),3′端删除387bp(129aa),表达产物具有甘露聚糖酶活性,这是本实验所找到的最小的且具有甘露聚糖酶活性的序列片段,即为源甘露聚糖酶基因序列长度的45.1%。
采用并联模式尝试构建复合酶表达体系,是一次新的策略,据现有资料来看,尚属首次。首先构建单质酶的复合表达载体,再通过热激转化法,将单质酶载体整合同一表达宿主中。将两种单质酶载体整合到同一宿主,获得了P-M、P-X、M-X表达体系,结果表明,一种单质酶的表达并不影响另外一种酶。将三种单质酶载体整合到同一宿主,获得的P-M-X表达体系同时分泌果胶酶、甘露聚糖酶、木聚糖酶三种酶,与现有生产用的菌株相比,果胶酶酶活相同,甘露聚糖酶酶活提高41.7%、木聚糖酶酶活提高了350%。脱胶实验表明,P -M-X的脱胶效果远远不如现在生产上所用的菌株。
The extracting of herbaceous fiber is a process to make cellulose apart from non-cellulose. The process is completed by enzyme produced by microbiology. The effect of bio-extracting of herbaceous fiber depends on the strain. Though there are various types microbiology that can secreting enzyme to degrade non-cellulose in the nature, a few kind of microbiology can be directly applied to extracting of herbaceous fiber. The key problem is how to improve the characters of the strain. The paper is on the key enzyme for bio-extracting of herbaceous fiber, including three parts: novel gene, lose expression and constructing compound enzyme expression system.
By comparison the clearing halos and the enzyme activity, CXJZ-166 was the strain of pectinase high, which was the most suitable strain for cloning pectinase gene. By PCR, a fragment of DNA were amplified from the the genome DNA of CXJZ-166.The PCR product were linked with pMD 18-T vector, transformed into E.coli JM109. Clear halos were made by positive clones in pectinase selecting plate. The DNA fragment is 877bp,coding 277aa, had been registered in Genbank under the accession number of EU597234.
Lose expression ofβ-mannanase had been tested systematically. The DNA sequence were shorten to 513bp (the full length 1134bp) in 3′end, accounting for 45% of the full length ,which hasβ-mannanase activity. 291bp had been deleted in 5′end, accounting for 25.6%, the fragment had the mannanase activity,. If some fragment ofβ-mannanase gene were deleted in two ends at the same time, 234bp in 5′and 387bp in 3′could be truncated. This was the most short fragment with mannanase activity in the paper, accounting for 45.1%.
Some kinds of enzyme were integrated into the same strain to secret kinds of enzyme simultaneously. Three kinds of two-enzyme strain were acquired in bio-extracting of herbaceous fiber,which were pectinase, mannanase and xylanase. Three kinds strain producing two kinds of enzyme had been acquired, which were Pectinase-Mannanase strain, Pectinase-Xylanase strain and Mannanase-Xylanase strain. P-M-X compound enzyme expression can secret pectinase,mannanase and xylanase. Mannanase and xylanase activity in fermented liquid of P-M-X were increased by 41.7% and 350% than the strain for production now.
引文
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