碱性纤维素酶产生菌的筛选、酶基因克隆及在大肠杆菌中的表达研究
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摘要
纤维素资源和纤维素酶是各国研究的热点领域,其中细菌产的碱性纤维素酶是研究的一个重要方面,碱性纤维素酶在洗涤、纺织、造纸、环保等行业都具有广阔的应用前景。
本文从碱性土样中,筛选获得20余株碱性纤维素酶产生菌株,经摇瓶复筛,得到一株酶活最高的嗜碱细菌Z-16。经16S rDNA序列比对和生理生化特征检测,初步鉴定Z-16属于芽孢杆菌属(Bacillus)。对粗酶液性质研究表明：Bacillussp.Z-16所产纤维素酶最适作用pH 7.0-7.5,在pH 6.5-9.0的范围有80%以上酶活,最适作用温度为50℃；该酶具有良好的pH和温度稳定性,在pH 3-10的条件下保存2h后,粗酶液仍能保持90%以上的酶活；Na+、K+和Mn2+对酶有明显的激活作用,Co2+、Hg2+、Cd2+和SDS抑制酶的活性,Zn2+和Cu2+使酶完全失活,而EDTA、Triton X-100和Tween 20对酶活无显著影响。
对Bacillus sp. Z-16的摇瓶产酶条件进行优化,最佳产酶条件为：碳源为CMC-Na,氮源为酵母粉,初始培养pH 9.0,培养基装液量10%,接种量3%,培养温度30℃,摇床转速120rpm,培养36 h。优化后,Bacillus sp. Z-16的纤维素酶酶活可达3.48 U／mL,是优化前的7.5倍。
通过对已发表的同源纤维素酶序列进行分析,设计引物,以Bacillus sp. Z-16基因组为模板,扩增得到含Z-16纤维素酶基因的DNA片段,经克隆测序,结果表明该酶基因ORF长2475 bp,编码824个氨基酸。对演推的蛋白序列分析表明,该酶N-端有30个氨基酸的信号肽,催化结构域属于糖苷水解酶第5家族,C端为CBM_17_28家族的纤维结合结构域。该酶去除信号肽的成熟酶计算分子量88,181 Da, pI值为4.19。该酶的催化结构域与日本投入应用的洗涤用碱性纤维素酶Cel K有72%的同源性。
将Z-16纤维素酶去除信号肽的全酶基因(gen 2)和催化结构域部分基因(gen3)分别克隆至pET-28a(+)表达载体,在E.coli BL21 (DE3)中进行高效表达。对重组酶Cel 2和Cel 3的胞外表达条件进行了优化,当培养液菌浓A600达到0.8时,加入0.8 mM的IPTG,37℃诱导16h后,Cel 2和Cel 3胞外酶活达到最高,分别为1.191 U／mL和9.307 U／mL,此时它们胞外比活分别为17.80 U／mg和45.13U／mg。
重组酶Cel 2和Cel 3的胞外粗酶液经超滤浓缩处理后,进行His6标签一步亲和层析,得到纯化的重组酶Cel 2和Cel 3,纯化倍数分别为3.2和2.5倍,比活分别达到63.5 U／mg和89.0 U／mg。随后对两种纯酶的酶学性质研究表明：重组全酶Cel 2的最适作用pH为7.5,在7.5-9.0的pH范围有90%以上酶活,最适温度50℃,均表现与粗酶液一致的特性；重组催化域Cel 3的最适作用pH为7.5,在6.5-8.0的pH范围有90%以上酶活,较Cel 2偏酸了一个pH单位,而Cel 3的最适作用温度提高至55-60℃；金属离子、EDTA及表面活性剂对Cel 2和Cel3的影响,均与对粗酶液的影响基本一致。此外,还测定了纯酶的Km和Vmax,Cel 2和Cel 3分别为0.105 mmol/L和0.900 mmol/L,0.05 mmol/L/min和0.280mmol/L/min。
综上所述,本研究首先通过筛选获得一个产pH和温度稳定性良好的碱性纤维素酶的嗜碱芽孢杆菌,对其产酶条件进行了优化,随后克隆了纤维素酶基因,实现了酶基因的异源表达,纯化了重组酶并研究了酶学特性,为研究碱性纤维素酶耐碱机理提供了材料,为实现碱性纤维素酶在工业领域的应用奠定了基础。
The study on cellulose resources and cellulases has become a current research hotspot and received intensive attention all over the world. Among the various types of cellulases widespread in nature, the bacterial alkaline cellulases are of great importance and extensively applied in detergent, textile, paper manufacturing, environmental protection, and many other industrial fields.
More than 20 bacteria strains producing alkaline cellulases were isolated from alkaline soil samples, among which, the alkalophilic bacterium Z-16, exhibited highest cellulase activity. Phylogenetic analysis based on 16S rDNA gene sequence together with part of physiological and biochemistry characteristics indicated that Z-16 belonged to the genus of Bacillus. The highest CMCase activity of the crude cellulase produced by Bacillus sp. Z-16 was obtained at 50℃and pH 7.0-7.5, and approx.80% of the activity was within pH 6.5-9.0. This enzyme showed excellent pH stability and thermostability. It retained more than 90% of its maximal activity for 2 h at pH 3-11. The activity of this enzyme was activated by Na+, K+ and Mn2+, but inhibited by Co2+, Hg2+, Cd2+ and SDS. Whereas Zn2+ and Cu2+ caused deactivation of this enzyme, but EDTA, Triton X-100 and Tween 20 had no significant effect on the activity.
The growth conditions that influenced the cellulase production of Bacillus sp. Z-16 were investigated and optimized. When CMC-Na was used as carbon source and yeast extract as nitrogen source, initial pH 9.0,10% (v/v) medium volume,3% (v/v) inoculum,30℃and shaken at 120 rpm for 36 h, the highest cellulase activity reached 3.48 U/mL, which was 7.5 times higher than its initial value.
Primers were designed based on the published sequences of cellulase genes from homologous strains. The cellulase gene fragment of Bacillus sp. Z-16 was amplified by using its chromosomal DNA as template and sequenced. Its ORF consists of 2475 nucleotides and encodes a protein composed of 824 amino acids. The calculated molecular weight was 88,181 daltons and predicted pI was 4.70. According to the analysis of its amino acids, this enzyme consists of a signal peptide (30 amino acids), a glycosyl hydrolase family five domain at its N terminus, and a CBM_17_28 cellulose binding domain at its C terminus. The catalytic domain of the cellulase shows 72% identity with Cel K, an alkaline cellulase applied in detergent industry in Japan.
The gene (gen 2) encoding for cellulase without signal peptide and the gene (gen 3) encoding for the catalytic domain were cloned and inserted into pET-28a(+), then expressed in E. coli BL21 (DE3), respectively. The condition for expressing the corresponding recombinant cellulase, named Cel 2 and Cel 3, was optimized, respectively.0.8 mM of IPTG was added when the cell density reached 0.8 of OD600 followed by 16 hours of cultivation at 37℃, the extracellular activities of Cel 2 and Cel 3 reached 1.191 U/mL and 9.307 U/mL, and the specific activities were 17.80 U/mg and 45.13 U/mg, respectively.
The extracellular Cel 2 and Cel 3 were concentrated by ultrafiltration. After the procedure of affinity chromatography, they were purified 3.2-fold and 2.5-fold with specific activities of 63.5 U/mg and 89.0 U/mg, respectively. The maximum CMCase activity of Cel 2 was measured at 50℃and pH 7.5, and 90% of the activity was obtained at pH 7.5-9.0, which was similar with wild-type enzyme. However, the maximum CMCase activity of Cel 3 was measured at 55-60℃and pH 7.5, and 90% of the activity at pH 6.5-8.0, which exhibited a acidic shift of 1.0 pH unit compared to Cel 2 and the wild-type enzyme. The effects of metal ions, EDTA and surfactants on the activity of Cel 2 and Cel 3 were the same as that on the wild-type enzyme. The Km and Vmax of purified Cel 2 and Cel 3 were also measured. The Km value of Cel 2 was 0.105 mmol/L, and 0.900 mmol/L for Cel 3. The Vmax of Cel 2 was 0.05 mmol/L/min and 0.280 mmol/L/min for Cel 3.
In this reaserch, an alkalophilic bacillus producing alkaline cellulase with excellent pH stability and thermostability was isolated. The gene encoding for the cellulase was cloned and expressed in E.coli. The recombinant cellulase and its CD domain were purified, and studied. This work not only provides a good material for the study of alkali-tolerrance mechanism of cellulase, but also benefits for the application of alkaline cellulase.
本文从碱性土样中,筛选获得20余株碱性纤维素酶产生菌株,经摇瓶复筛,得到一株酶活最高的嗜碱细菌Z-16。经16S rDNA序列比对和生理生化特征检测,初步鉴定Z-16属于芽孢杆菌属(Bacillus)。对粗酶液性质研究表明：Bacillussp.Z-16所产纤维素酶最适作用pH 7.0-7.5,在pH 6.5-9.0的范围有80%以上酶活,最适作用温度为50℃；该酶具有良好的pH和温度稳定性,在pH 3-10的条件下保存2h后,粗酶液仍能保持90%以上的酶活；Na+、K+和Mn2+对酶有明显的激活作用,Co2+、Hg2+、Cd2+和SDS抑制酶的活性,Zn2+和Cu2+使酶完全失活,而EDTA、Triton X-100和Tween 20对酶活无显著影响。
对Bacillus sp. Z-16的摇瓶产酶条件进行优化,最佳产酶条件为：碳源为CMC-Na,氮源为酵母粉,初始培养pH 9.0,培养基装液量10%,接种量3%,培养温度30℃,摇床转速120rpm,培养36 h。优化后,Bacillus sp. Z-16的纤维素酶酶活可达3.48 U／mL,是优化前的7.5倍。
通过对已发表的同源纤维素酶序列进行分析,设计引物,以Bacillus sp. Z-16基因组为模板,扩增得到含Z-16纤维素酶基因的DNA片段,经克隆测序,结果表明该酶基因ORF长2475 bp,编码824个氨基酸。对演推的蛋白序列分析表明,该酶N-端有30个氨基酸的信号肽,催化结构域属于糖苷水解酶第5家族,C端为CBM_17_28家族的纤维结合结构域。该酶去除信号肽的成熟酶计算分子量88,181 Da, pI值为4.19。该酶的催化结构域与日本投入应用的洗涤用碱性纤维素酶Cel K有72%的同源性。
将Z-16纤维素酶去除信号肽的全酶基因(gen 2)和催化结构域部分基因(gen3)分别克隆至pET-28a(+)表达载体,在E.coli BL21 (DE3)中进行高效表达。对重组酶Cel 2和Cel 3的胞外表达条件进行了优化,当培养液菌浓A600达到0.8时,加入0.8 mM的IPTG,37℃诱导16h后,Cel 2和Cel 3胞外酶活达到最高,分别为1.191 U／mL和9.307 U／mL,此时它们胞外比活分别为17.80 U／mg和45.13U／mg。
重组酶Cel 2和Cel 3的胞外粗酶液经超滤浓缩处理后,进行His6标签一步亲和层析,得到纯化的重组酶Cel 2和Cel 3,纯化倍数分别为3.2和2.5倍,比活分别达到63.5 U／mg和89.0 U／mg。随后对两种纯酶的酶学性质研究表明：重组全酶Cel 2的最适作用pH为7.5,在7.5-9.0的pH范围有90%以上酶活,最适温度50℃,均表现与粗酶液一致的特性；重组催化域Cel 3的最适作用pH为7.5,在6.5-8.0的pH范围有90%以上酶活,较Cel 2偏酸了一个pH单位,而Cel 3的最适作用温度提高至55-60℃；金属离子、EDTA及表面活性剂对Cel 2和Cel3的影响,均与对粗酶液的影响基本一致。此外,还测定了纯酶的Km和Vmax,Cel 2和Cel 3分别为0.105 mmol/L和0.900 mmol/L,0.05 mmol/L/min和0.280mmol/L/min。
综上所述,本研究首先通过筛选获得一个产pH和温度稳定性良好的碱性纤维素酶的嗜碱芽孢杆菌,对其产酶条件进行了优化,随后克隆了纤维素酶基因,实现了酶基因的异源表达,纯化了重组酶并研究了酶学特性,为研究碱性纤维素酶耐碱机理提供了材料,为实现碱性纤维素酶在工业领域的应用奠定了基础。
The study on cellulose resources and cellulases has become a current research hotspot and received intensive attention all over the world. Among the various types of cellulases widespread in nature, the bacterial alkaline cellulases are of great importance and extensively applied in detergent, textile, paper manufacturing, environmental protection, and many other industrial fields.
More than 20 bacteria strains producing alkaline cellulases were isolated from alkaline soil samples, among which, the alkalophilic bacterium Z-16, exhibited highest cellulase activity. Phylogenetic analysis based on 16S rDNA gene sequence together with part of physiological and biochemistry characteristics indicated that Z-16 belonged to the genus of Bacillus. The highest CMCase activity of the crude cellulase produced by Bacillus sp. Z-16 was obtained at 50℃and pH 7.0-7.5, and approx.80% of the activity was within pH 6.5-9.0. This enzyme showed excellent pH stability and thermostability. It retained more than 90% of its maximal activity for 2 h at pH 3-11. The activity of this enzyme was activated by Na+, K+ and Mn2+, but inhibited by Co2+, Hg2+, Cd2+ and SDS. Whereas Zn2+ and Cu2+ caused deactivation of this enzyme, but EDTA, Triton X-100 and Tween 20 had no significant effect on the activity.
The growth conditions that influenced the cellulase production of Bacillus sp. Z-16 were investigated and optimized. When CMC-Na was used as carbon source and yeast extract as nitrogen source, initial pH 9.0,10% (v/v) medium volume,3% (v/v) inoculum,30℃and shaken at 120 rpm for 36 h, the highest cellulase activity reached 3.48 U/mL, which was 7.5 times higher than its initial value.
Primers were designed based on the published sequences of cellulase genes from homologous strains. The cellulase gene fragment of Bacillus sp. Z-16 was amplified by using its chromosomal DNA as template and sequenced. Its ORF consists of 2475 nucleotides and encodes a protein composed of 824 amino acids. The calculated molecular weight was 88,181 daltons and predicted pI was 4.70. According to the analysis of its amino acids, this enzyme consists of a signal peptide (30 amino acids), a glycosyl hydrolase family five domain at its N terminus, and a CBM_17_28 cellulose binding domain at its C terminus. The catalytic domain of the cellulase shows 72% identity with Cel K, an alkaline cellulase applied in detergent industry in Japan.
The gene (gen 2) encoding for cellulase without signal peptide and the gene (gen 3) encoding for the catalytic domain were cloned and inserted into pET-28a(+), then expressed in E. coli BL21 (DE3), respectively. The condition for expressing the corresponding recombinant cellulase, named Cel 2 and Cel 3, was optimized, respectively.0.8 mM of IPTG was added when the cell density reached 0.8 of OD600 followed by 16 hours of cultivation at 37℃, the extracellular activities of Cel 2 and Cel 3 reached 1.191 U/mL and 9.307 U/mL, and the specific activities were 17.80 U/mg and 45.13 U/mg, respectively.
The extracellular Cel 2 and Cel 3 were concentrated by ultrafiltration. After the procedure of affinity chromatography, they were purified 3.2-fold and 2.5-fold with specific activities of 63.5 U/mg and 89.0 U/mg, respectively. The maximum CMCase activity of Cel 2 was measured at 50℃and pH 7.5, and 90% of the activity was obtained at pH 7.5-9.0, which was similar with wild-type enzyme. However, the maximum CMCase activity of Cel 3 was measured at 55-60℃and pH 7.5, and 90% of the activity at pH 6.5-8.0, which exhibited a acidic shift of 1.0 pH unit compared to Cel 2 and the wild-type enzyme. The effects of metal ions, EDTA and surfactants on the activity of Cel 2 and Cel 3 were the same as that on the wild-type enzyme. The Km and Vmax of purified Cel 2 and Cel 3 were also measured. The Km value of Cel 2 was 0.105 mmol/L, and 0.900 mmol/L for Cel 3. The Vmax of Cel 2 was 0.05 mmol/L/min and 0.280 mmol/L/min for Cel 3.
In this reaserch, an alkalophilic bacillus producing alkaline cellulase with excellent pH stability and thermostability was isolated. The gene encoding for the cellulase was cloned and expressed in E.coli. The recombinant cellulase and its CD domain were purified, and studied. This work not only provides a good material for the study of alkali-tolerrance mechanism of cellulase, but also benefits for the application of alkaline cellulase.
引文
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