摘要
纤维类物质是地球上数量最大的可再生资源,植物每年通过光合作用产生多达10-50亿吨的纤维类物质。目前大部分纤维类物质未能充分开发利用,造成了巨大的浪费和严重的环境污染。运用微生物对纤维类物质进行充分的酶解作用,可将其转化成糖、酒精或化工原料等,对缓解全球能源危机、食品紧张以及环境污染有着重大意义。
本文在黄山生态林腐木筛选出一株纤维素酶产生菌的基础上,对其在分类学上地位、纤维素酶编码基因进行系统分析,并依此构建纤维素酶基因工程菌。主要研究成果如下：
1)利用CMC-Na刚果红平板筛选法,从黄山生态林腐木中分离等到一株纤维素酶产生菌,通过原生质体紫外诱变选育获得一株纤维素酶高产菌株,命名为TP-02；其纤维素酶的滤纸酶活及内切葡聚糖苷酶酶活分别达到7.56 IU/mL、28.5IU/mL,其发酵时间与目前研究较多的里氏木霉相比大约提前36h。
2)以分子生物学鉴定方法为依据,辅以菌种的形态学鉴定对突变株TP-02进行菌种鉴定。研究结果表明突变株TP-02隶属于接合菌亚门、藻状菌纲、毛霉目、毛霉科、根霉属、匍枝根霉亚种点头根霉(Zygomycotina, Phycomycetes, Mucorales, Mucoraceae, Rhizopus stolonifer var. reflexus)。
3)利用匍枝根霉亚种点头根霉TP-02的mRNA模板,在逆转录酶的作用下,以Oligod(T)18引导合成cDNA第一链,同时在GenBank上搜索该菌株近缘属种的内切葡聚糖苷酶基因序列并设计引物,进行PCR扩增,序列经GenBank数据库比对分析为新的内切葡聚糖苷酶编码序列,提交GenBank,获得序列登录号为FJ807269。将扩增产物与原核表达载体pET20b连接构建表达载体pET20b-EG1,并转染大肠杆菌BL21(DE3)。重组菌发酵36h后,经IPTG诱导可达到最大值0.715IU/mL;发酵产物经纯化后,经SDS-PAGE检测,可达到一相对分子量大约为40kDa的目的片段。
4)利用匍枝根霉亚种点头根霉TP-02的mRNA模板,在逆转录酶的作用下,以锚定引物Oligo (dT)18 Anchor Primer:5'-(GA)10 CTCGAGCGGCCGC(T)18 V-3'为引导,合成双链全长cDNA,同时加上含有酶切位点的接头,经酶切后与毕赤酵母表达载体pPIC9K相连构建表达质粒,转染毕赤酵母GS115感受态细胞。将所获得cDNA文库利用CMC刚果红平板筛选法,获得阳性单克隆,酶切测序,获得3个不同的序列,经比对分析其中一个序列与已提交的序列同源率达到100%,其余序列同源率较低,可能为新的内切葡聚糖苷酶编码序列,提交GenBank,获得序列登录号分别为HM043656和HM043657；将序列所对应的菌株进行发酵分析,经甲醇诱导培养60h后,其内切葡聚糖苷酶酶活可达到最大值分别为1.15IU/mL、1.86IU/mL以及1.56IU/mL.经SDS-PAGE检测获得相对分子量大约为40kDa、44 kDa、46kDa 3条带对应于序列登录号为FJ807269、HM043656、HM043657.
Cellulose is the most abundant renewable nature product in the biosphere. Annual production of cellulose is estimated to be 10-50×109 tons. Presently, most of cellulose are wasted and pollute environment. So it has great realistic meaning for human being to solve energy crisis, food shortage and environment pollution that using cellulose produced by microorganism transform cellulose to energy, food and chemical.
During this paper, a cellulase-producing strain was screened from ecological forest in Huangshan, on basis of that, taxonomy position, and the construction of cellulase gene had been studied, so as to construct genetically cellulase engineering strain. The major results are as follows:
A strain with high activity of cellulase was sparated from rotten wood of ecological forest by the mothed of CMC-congon red plate screening. During the mothed of the mutagenesis on protoplast by Ultraviolet, a high activity of cellulase strain had been obtained and named TP-02; the Filter Paper Activity (FPA) and Endoglucanase (CMCase) of the strain could be achieved 7.56 IU/mL and 28.5IU/mL respectively, the fermentation time advanced 36h compared with the current study strain Trichoderma reesei QM9414.
The mutant TP-02 was identified by utilization of the methods of both molecular biological identification and morphological identification. The results of that demonstration that the strain used in this paper, belong to Zygomycotina, Phycomycetes, Mucorales, Mucoraceae, Rhizopus stolonifer var.reflexus.
The first stand cDNA synthesised by using the Rhizopus stolonifer var.reflexus mRNA template, and guiding with primer Oligod (T) 18, under the condition of reverse transcriptase. The endo-glucanase gene sequences of the related species were searched to design the primers by softares to amplify the first stand cDNA. The sequence identified as new gene encoding Endo-glucanase by the program of Blast in NCBI, and submitted to GenBanK, and a receiving number of the sequence FJ807269 was obtained. Plasmid pET20b-EG1 was constructed by ligation the PCR products to the prokaryotic expression vector pET20b, and then transfected it into E. coli BL21 (DE3). After 36h fermentation and induction by IPTG, the recombinant bacteria's Endo-glucanase activity could achieve the peak of 0.715IU/mL. a band with molecular weight about 40 kDa could be detected by SDS-PAGE of the purified fermentation product.
Under the condition of reverse transcriptase, double-stranded length cDNA was synthesized by utilizing Rhizopus stolonifer var.reflexus TP-02 mRNA template, and guiding with Anchor primer 5'-(GA) 10 CTCGAGCGGC CGC (T) 18 V-3'. Then an adaptor containing EcoR I site was joined with that by the ligation kit. After that, the double-stranded cDNA with both EcoR I and Not I site was digested by restriction endonuculease Not I and linked into double digested vector pPIC9K. After that, the plasimid was transfected into Pichia pastoris GS115 competent cells to construct cDNA library. The positive clones were sceened by the mothed of CMC-congon red plate from the cDNA library. Three different sequences were obtained after double digested and sequenced the positive clones. One sequence showed 100% Similarity with the sequence FJ807269, others were new gene encoding Endo-gluancanase, after alignment analysis, and submitted to GenBank with the accession number:HM043656 and HM043657. After 60h fermentation and induction by methanol, the recombinant Yeasts' Endo-glucanase activity could achieve the peak of 1.15 IU/mL 1.86IU/mL and 1.56IU/mL. Three bands with molecular weight about 40 kDa 44 kDa,46kDa could be detected by SDS-PAGE of the purified fermentation product corresponding to the sequence of FJ807269, HM043656, and HM043657.
引文
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