普鲁兰短梗霉HN2-3菌株碱性蛋白酶的纯化、基因克隆、表达、表面展示及生产活性肽的应用研究
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摘要
从分离自海水、海泥及海洋生物内脏及海生植物表面的400余株酵母菌中分离到5株产蛋白酶的海洋酵母，能在酪蛋白双层平板上形成明显的透明圈，它们分别是HN2-3、N13d、N13C、Mb5和HN3-2。通过酵母鉴定的常规方法并结合分子生物学方法，鉴定出HN2-3、N13d、N13C与HN3-24株菌属于普鲁兰短梗霉(Aureobasidiumpullulans)，Mb5菌株属于解脂亚罗酵母(Yarrowia lipolytica)。来自菌株HN2-3所产蛋白酶酶活性及酶解活性肽产物的ACE(AngiotensinI-converting enzyme)抑制活性最高。来自菌株HN2-3的蛋白酶命名为ALP2。
利用层析柱Sephadex G-75与阴离子交换柱DEAE Sepharose Fast Flow纯化了菌株HN2-3的胞外碱性蛋白酶ALP2，纯化2．34倍。通过SDS-PAGE凝胶电泳，得到的ALP2蛋白条带分子量为33．0 kDa，最适作用温度为52℃。Mn~(2+)、Mg~(2+)和Na~+离子浓度在5 mM时对纯化蛋白酶酶活有激活作用，Zn~(2+)、Ca~(2+)、K~+和Li~+对酶活的影响不大；而当存在Fe~(2+)、Fe~(3+)、Cu~(2+)、Co~(2+)、Ag~+、和Hg~(2+)离子时，蛋白酶活明显降低。苯甲基磺酰氟(PMSF)强烈抑制蛋白ALP2的活性，乙二胺四乙酸(EDTA)与碘乙酸微弱抑制蛋白酶的活性。利用纯化的酶酶解砺虾蛋白，螺旋藻蛋白(Arthospiraplatensis)，酵母N3C(Yarrowia，lipolytica)和YA03a(Hansenia sporauvarum)蛋白，来自蛎虾蛋白的水解物ACE抑制活性与抗氧化活性最高，分别达到88．3％与47．3％。这说明纯化的ALP2蛋白酶在食品和医药方面具有良好的前景。
利用简并PCR、反向PCR与RT-PCR获得了蛋白酶ALP2基因的DNA序列与cDNA序列。基因ALP2的DNA序列为1354 bp，在NCBI(http：／／www．ncbi．nlm．nih．gov／)的登陆号为EU331441。ALP2基因的开放阅读框(ORF)1248 bp，在NCBI上的登陆号为EU224431，编码415个氨基酸，推导分子量为42．9 kDa。基因ALP2具有两个内含子，分别为54bp和52 bp。通过分子生物学软件分析，基因ALP2前18个氨基酸为信号肽序列，此基因编码的蛋白具有丝氨酸活性位点与组氨酸活性位点，等电点为6．44。利用质粒pINA1317对基因进行表达验证，发现转化子能在牛奶-PPB双层平板上形成明显的透明圈。转化子的发酵上清液的蛋白酶活性也进行了检测，结果说明克隆到的基因ALP2确实为蛋白酶基因，而且能在Y.lipolytica中进行表达，并分泌到细胞外。
利用本实验室构建的表面展示质粒pINA1317-YICPW110对蛋白酶基因ALP2进行了表面展示，转化子能在牛奶双层平板上形成明显的透明圈。表面展示的蛋白酶酶解不同的蛋白源能够产生活性肽，发现酶解金黄色隐球酵母G7a(Cryptococcus aureus)的细胞蛋白所产活性肽的ACE抑制活性最高，酶解螺旋藻蛋白所产活性肽的抗氧化活性最高。这是首次关于利用表面展示蛋白酶进行活性肽的生产的报道。
将表面展示的蛋白酶进行了酶学特性研究，发现与菌株HN2-3所产的野生型蛋白酶相比，最适作用温度有所降低，温度稳定性增强；另外发现表面展示的蛋白酶对金属离子的亲和力降低。
Over 400 yeast strains were isolated from seawater, sediments, bowels of marine organisms and surface of thalassophyte, we found 5 strains( HN2-3, N13d, N13C, Mb5 and HN3-2) among them could form clear zone around the colonies on the double plates with 2.0 % casein. Strains HN2-3, N13d, N13C and HN3-2 were identified as Aureobasidium pullulans and strain Mb5 was identified as Yarrowia lipolytica using morphology, physiology and molecular methods. Strain HN2-3 had highest protease activity. Peptides in the hydrolysate produced by the proteases from strain HN2-3 had the highest angiotensin I-converting-enzyme (ACE)-inhibitory activity and the protease from HN2-3 was named as ALP2.
The extracellular alkaline protease ALP2 in the supernatant of cell culture of the marine yeast A. pullulans HN2-3 was purified to homogeneity with a 2.34-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography(Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamidegel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 33.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 52℃, respectively. The enzyme was activated by Mg~(2+)、Na~+and Mn~(2+) (at a concentration of 5.0 mM) and inhibited by Fe~(2+)、Fe~(3+)、Cu~(2+)、Co~(2+)、Ag~+ and Hg~(2+). Zn~(2+)、Ca~(2+)、K~+ and Li~+ has no effect on the protease activity. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, and weakly inhibited by EDTA and iodoacetic acid. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strainsN3C (Y. lipolytica) and YA03a (Hansenia sporauvarum) was digested by the purified alkaline protease, the highest values of ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were 88.3% and 47.3 %, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceuticalindustries.
The alkaline protease structural gene (ALP2 gene) was obtained from both the genomic DNA and cDNA of A. pullulans HN2-3 by degenerate PCR, inverse PCR and RT-PCR. DNA sequence of the ALP2 gene ( accession number: EU331441) was 1354bp. An open reading frame of 1248 bp (accession number: EU224431 ) encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. Eighteen amino acid at the beginning of the gene was signal peptide. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The protease ALP2 gene was expressed by the plasmid pINA1317.The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2.0 % milk and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP2 gene was protease gene which was expressed in Y. lipolytica and the expressed alkaline protease was secreted into the medium.
When the cDNAALP2 gene was cloned into multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Y. lipolytica. the protease displaying cells could form the clear zone on the double plate containing milk protein. The alkaline protease displaying cells were also found to be able to produce bioactive peptides from different sources of proteins. The produced peptide from single cell protein of marine yeast strain G7a had the highest ACE inhibitory activity while the produced peptide from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides.
The properties of the surface displayed protease were examined. It was found that the optimal temperature of the displayed alkaline protease was lower than that of the free alkaline protease. However, the thermal stability of the displayed protease was enhanced compared to that of the free protease. The pH stability of the displayed protease was different from that of the free protease. The results reveal that the displayed alkaline protease exhibited a lower affinity to azocasein than the free one.
利用层析柱Sephadex G-75与阴离子交换柱DEAE Sepharose Fast Flow纯化了菌株HN2-3的胞外碱性蛋白酶ALP2，纯化2．34倍。通过SDS-PAGE凝胶电泳，得到的ALP2蛋白条带分子量为33．0 kDa，最适作用温度为52℃。Mn~(2+)、Mg~(2+)和Na~+离子浓度在5 mM时对纯化蛋白酶酶活有激活作用，Zn~(2+)、Ca~(2+)、K~+和Li~+对酶活的影响不大；而当存在Fe~(2+)、Fe~(3+)、Cu~(2+)、Co~(2+)、Ag~+、和Hg~(2+)离子时，蛋白酶活明显降低。苯甲基磺酰氟(PMSF)强烈抑制蛋白ALP2的活性，乙二胺四乙酸(EDTA)与碘乙酸微弱抑制蛋白酶的活性。利用纯化的酶酶解砺虾蛋白，螺旋藻蛋白(Arthospiraplatensis)，酵母N3C(Yarrowia，lipolytica)和YA03a(Hansenia sporauvarum)蛋白，来自蛎虾蛋白的水解物ACE抑制活性与抗氧化活性最高，分别达到88．3％与47．3％。这说明纯化的ALP2蛋白酶在食品和医药方面具有良好的前景。
利用简并PCR、反向PCR与RT-PCR获得了蛋白酶ALP2基因的DNA序列与cDNA序列。基因ALP2的DNA序列为1354 bp，在NCBI(http：／／www．ncbi．nlm．nih．gov／)的登陆号为EU331441。ALP2基因的开放阅读框(ORF)1248 bp，在NCBI上的登陆号为EU224431，编码415个氨基酸，推导分子量为42．9 kDa。基因ALP2具有两个内含子，分别为54bp和52 bp。通过分子生物学软件分析，基因ALP2前18个氨基酸为信号肽序列，此基因编码的蛋白具有丝氨酸活性位点与组氨酸活性位点，等电点为6．44。利用质粒pINA1317对基因进行表达验证，发现转化子能在牛奶-PPB双层平板上形成明显的透明圈。转化子的发酵上清液的蛋白酶活性也进行了检测，结果说明克隆到的基因ALP2确实为蛋白酶基因，而且能在Y.lipolytica中进行表达，并分泌到细胞外。
利用本实验室构建的表面展示质粒pINA1317-YICPW110对蛋白酶基因ALP2进行了表面展示，转化子能在牛奶双层平板上形成明显的透明圈。表面展示的蛋白酶酶解不同的蛋白源能够产生活性肽，发现酶解金黄色隐球酵母G7a(Cryptococcus aureus)的细胞蛋白所产活性肽的ACE抑制活性最高，酶解螺旋藻蛋白所产活性肽的抗氧化活性最高。这是首次关于利用表面展示蛋白酶进行活性肽的生产的报道。
将表面展示的蛋白酶进行了酶学特性研究，发现与菌株HN2-3所产的野生型蛋白酶相比，最适作用温度有所降低，温度稳定性增强；另外发现表面展示的蛋白酶对金属离子的亲和力降低。
Over 400 yeast strains were isolated from seawater, sediments, bowels of marine organisms and surface of thalassophyte, we found 5 strains( HN2-3, N13d, N13C, Mb5 and HN3-2) among them could form clear zone around the colonies on the double plates with 2.0 % casein. Strains HN2-3, N13d, N13C and HN3-2 were identified as Aureobasidium pullulans and strain Mb5 was identified as Yarrowia lipolytica using morphology, physiology and molecular methods. Strain HN2-3 had highest protease activity. Peptides in the hydrolysate produced by the proteases from strain HN2-3 had the highest angiotensin I-converting-enzyme (ACE)-inhibitory activity and the protease from HN2-3 was named as ALP2.
The extracellular alkaline protease ALP2 in the supernatant of cell culture of the marine yeast A. pullulans HN2-3 was purified to homogeneity with a 2.34-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography(Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamidegel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 33.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 52℃, respectively. The enzyme was activated by Mg~(2+)、Na~+and Mn~(2+) (at a concentration of 5.0 mM) and inhibited by Fe~(2+)、Fe~(3+)、Cu~(2+)、Co~(2+)、Ag~+ and Hg~(2+). Zn~(2+)、Ca~(2+)、K~+ and Li~+ has no effect on the protease activity. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, and weakly inhibited by EDTA and iodoacetic acid. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strainsN3C (Y. lipolytica) and YA03a (Hansenia sporauvarum) was digested by the purified alkaline protease, the highest values of ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were 88.3% and 47.3 %, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceuticalindustries.
The alkaline protease structural gene (ALP2 gene) was obtained from both the genomic DNA and cDNA of A. pullulans HN2-3 by degenerate PCR, inverse PCR and RT-PCR. DNA sequence of the ALP2 gene ( accession number: EU331441) was 1354bp. An open reading frame of 1248 bp (accession number: EU224431 ) encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. Eighteen amino acid at the beginning of the gene was signal peptide. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The protease ALP2 gene was expressed by the plasmid pINA1317.The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2.0 % milk and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP2 gene was protease gene which was expressed in Y. lipolytica and the expressed alkaline protease was secreted into the medium.
When the cDNAALP2 gene was cloned into multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Y. lipolytica. the protease displaying cells could form the clear zone on the double plate containing milk protein. The alkaline protease displaying cells were also found to be able to produce bioactive peptides from different sources of proteins. The produced peptide from single cell protein of marine yeast strain G7a had the highest ACE inhibitory activity while the produced peptide from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides.
The properties of the surface displayed protease were examined. It was found that the optimal temperature of the displayed alkaline protease was lower than that of the free alkaline protease. However, the thermal stability of the displayed protease was enhanced compared to that of the free protease. The pH stability of the displayed protease was different from that of the free protease. The results reveal that the displayed alkaline protease exhibited a lower affinity to azocasein than the free one.
引文
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