分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列,将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ３１酶切,Ｔ４ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒,将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶,酶活测定结果表明,基因表达水平比用原有的启动子高１－２倍,蛋白质分泌率在８４－９６％之间。     

分泌载体pUS186的构建及地衣杆菌α—淀粉酶基因在枯草杆菌中的表…

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克枯草杆菌染色体的启动子和信号肽序列。将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ ３１酶切,Ｔ４ ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ｉ     

双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。     

枯草杆菌碱性蛋白酶基因诱导表达载体的构建

以PCR方法扩增sacB基因的启动子-信号肽序列(称为sacR),将其与枯草芽孢杆菌碱性蛋白酶的前肽-成熟酶基因连接后克隆入载体pUBH,构建了含碱性蛋白酶基因的分泌型诱导表达载体pUBS,将其转化枯草芽孢杆菌DB403后,获得基因工程菌DB403(pUBS)。碱性蛋白酶基因在sacR的调控和蔗糖的诱导下实现了表达分泌,获得了具生物学活性的碱性蛋白酶。     

枯草杆菌脂肪酶基因在大肠杆菌中的诱导分泌表达

借助生物信息学对已克隆的枯草杆菌脂肪酶LipB2全长基因序列进行比对分析。结果显示该脂肪酶基因全长635bp,编码包括31个氨基酸分泌型信号肽在内的211个氨基酸,与NCBIGenBank中已报道的枯草杆菌属脂肪酶核苷酸序列有94.0%的一致性。将该基因克隆到pET-28a(+)表达载体上,转化大肠杆菌BL21(DE3),利用枯草杆菌脂肪酶的信号肽序列进行了分泌表达。SDS-PAGE电泳显示分泌表达的脂肪酶分子质量约为21kD。对表达条件优化后,在30℃、大肠杆菌菌液OD600值为1.8、乳糖诱导浓度为1.5mM、摇瓶发酵10h后大肠杆菌分泌表达26.0U/mL重组脂肪酶,相比较IPTG的诱导,既实现了脂肪酶的高效表达,又节省了成本。     

一种新型酿酒酵母附加型分泌表达载体的构建

用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列(INU),将其嵌入酵母附加型表达质粒pYES2,得到一套新型的分泌表达载体pYES2I,pYES2Ⅱ,pYES2Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因(ASN)和短芽孢杆菌α乙酰乳酸脱羧酶(ALDC)基因,连接到INU下游,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株INVScⅠ中表达,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明,重组酵母菌株在没有选择压力的条件下连续接种培养100h,未发现重组质粒的不稳定性。     

大肠杆菌、枯草杆菌穿梭表达载体的构建及改造

将大肠杆菌的复制子rep和多克隆位点克隆到枯草杆菌质粒pGDV1的骨架上,即得到大肠杆菌枯草杆菌牙梭庾粒载俸pGDVM。在pGDVM上进行载体表达元件的构建,先后将P59启动子、核糖体结合位点SD和终止子克隆到pGDVM上得到穿梭表达载体GJ01。以β-半乳糖苷酶基因（bga）作为报告基因检测载体的表达活性,在大肠杆菌和枯草杆菌中β-半乳糖苷酶（Bga）酶活性最高达到75.3和83．2个密勒单位,表明所构建的表达载体具有较强的表达能力。以核糖体结合位点（SD、SD3、SD4和SD5）代替表达载体GJ01-bga中的SD,对载体进行改造。所构建的GJD2-bga在大肠杆菌中的最大酶活性为253．8个密勒单位,G]D5-bga在枯草杆菌中的最大酶活性为135．4个密勒单位,表明所构建的载体具有较强的表达活性。由此可以得出不同的SD序列及其与起始密码子的距离不同程度地影响mRNA的翻译效率。     

枯草杆菌中性蛋白酶基因在大肠杆菌中的表达

蛋白酶是枯草杆菌(Bacillus subtilis)产生的具有重要工业价值的水解酶。对蛋白酶基因的分离与高效率表达一直是基因工程研究领域的重要内容之一^[1-4]。蛋白酶基因的筛选可采用不同的方法,如“免疫法”、“DNA 杂交法”、“遗传互补法”等。大肠杆菌(Escherichia coli)是基因工程中最常用的宿主菌, 若能以E．Coli作为筛选蛋白酶基因的宿主苗,那么使用E．Coli的常规载体,便可直接获得完整的蛋白 酶基因。枯草杆菌的蛋白酶基因能否在大肠杆菌中表达．则是实现这一目标的关键。Koide等人^[5]报道过枯草杆菌的胞内丝氨酸蛋白酶基因在大肠杆菌中的表达。转化细胞在含有脱脂牛奶的平板上可产生十分微弱的水解圈。Ikeraara等人^[6]将Subtilisin E(枯草杆菌蛋白酶E)插人大肠杆菌的表达载体,具有活性的Subtilisin E便可分泌到大肠杆菌的细胞周质中。吴汝平撰文指出^[7]。克隆的枯草杆菌蛋白酶基因不能在大肠杆菌中表达。是因为大肠杆菌不能转录枯草杆菌的促使生长调节基因。Wang等人^[8]则认为,在大肠杆菌中观察不到野生型的中性蛋白酶基因E(nprE)的表达。是因为nprE的表达产物对大肠杆菌有致死作用．除去该基因上的核糖体结合位点,nprE便能在大肠杆菌中低水平表达,并能将表达产 物分泌至胞外。由上可知．枯草杆菌的蛋白酶基因能否在大肠杆菌中表达以及表达的位置仍然是一个众说纷纭的问题,这一问题也正是能否用大肠杆菌作为宿主菌筛选蛋白酶基因的关键。     

枯草杆菌蛋白酶E的蛋白质工程

用定点突变和随机突变的方法,对枯草杆菌碱性蛋白酶E基因进行改造。突变后的基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DBl04中进行表达,得到突变种的碱性蛋白酶．它们的突变位点分别是(M222A)、(M222A、N118S)、(M222A、N118S、Q103R)、(M222A、N118S、Q103R、D60N)。各突变种酶的性质测定 结果表明．M222A突变使酶抗氧化,N118S突变使酶增加热稳定性,Q103R和D60N突变虽然能增加酶的比活,但使酶的热稳定性大大下降,尤其是D60N突变使酶变得极不稳定。野生型碱性蛋白酶与(M222A)突变种的等电点均为8．92．而M222A,N118S)。(M222A,N118S ,Ql03R)和(M222A,118S．Q103R,D60N)突变酶分别为8.88．9.10和9．17。用Nsuc-AAPF-pNA作为底物时酶反应景适pH值为7.5～9.5,而用酪蛋白底物时最适pH值为10～12。     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。     

Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides

Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.Gram-positive bacteria of the genus Bacillus are industrially well-established microorganisms for the production of extracellular proteins. Due to the availability of relatively cheap large-scale production systems combined with the ability of bacteria to secrete up to 20 to 25 g/liter of a target protein into the growth medium, about 60% of commercially available enzymes are presently produced in Bacillus species (14, 28).The closely related species Bacillus subtilis and Bacillus licheniformis are widely used as production hosts on an industrial scale, and, in contrast to the well-known production species Escherichia coli, they are free of endotoxin and have GRAS (generally regarded as safe) status. The complete genome sequences of strains B. subtilis 168 (1, 18) and B. licheniformis DSM13 (isogenic to ATCC 14580) (26, 32) are available, greatly facilitating the construction of improved production strains.The Sec pathway constitutes the main secretion pathway in B. subtilis and B. licheniformis. Proteins secreted via the Sec pathway are initially synthesized with an N-terminal hydrophobic signal peptide (SP) consisting of a positively charged N domain followed by a longer, hydrophobic H domain and a C domain consisting of three amino acids which form the signal peptidase recognition site (35). Targeting of a secreted protein to the membrane, the translocation process itself, and subsequent processing by a signal peptidase represent the major bottlenecks for efficient translocation and thus production of heterologous proteins (20).SPs play a crucial role in the efficient translocation of secretory proteins by the Sec machinery. They interact with the SecA protein, the signal recognition particle (SRP), and the signal peptidase (16, 30). The interaction between the SP and the mature protein is known to influence protein export as well (9, 16, 17). Therefore, the choice of an efficient signal peptide for any given target protein is of utmost importance, and several approaches to identify efficient SPs for different target proteins were taken (2, 4, 6, 15, 21, 38).Among the huge number of enzymes produced on a large scale by Bacillus species, proteases are important for diverse industrial applications (25), with subtilisins being used as additives in household detergents (22, 28). We have chosen as a model for secretion optimization the subtilisin “Bacillus protease novo type” (BPN′) from Bacillus amyloliquefaciens ATCC 23844, a well-known enzyme belonging to the alkaline serine proteases (5).We present a novel approach to improve the extracellular production of this protease using different Bacillus host strains. A total number of 393 SPs were fused to the target protein, with 173 SPs originating from B. subtilis (termed homologous SPs) and 220 SPs from B. licheniformis DSM13 (termed heterologous SPs). The fusion constructs were cloned and expressed in B. subtilis, and the resulting library was screened for extracellular protease activity.     

中性蛋白酶基因诱导型表达分泌载体的构建

利用PCR方法分别扩增出sacB基因的启动子-信号肽序列（sacR）和枯草芽孢杆菌中性蛋白酶的前肽-成熟肽序列,将两者连接后克隆入载体pHP13中,构建了含有中性蛋白酶基因的诱导型表达分泌载体pHP13SN,再将其转化入枯草杆菌DB104,获得基因工程菌DB104(pHP13SN)。中性蛋白酶基因在蔗糖的诱导和sacR的调控下实现了分泌表达,并获得了具有生物学活性的中性蛋白酶。     

Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis

Streptomyces naraensis was inoculated into 100 ml of culture broth, containing 50 µCi of ⁶⁵Zn, diluted with ZnCl₂ solution to make 10^-4 m Zn²⁺ ion, at 27°C for 5 days with shaking. ⁶⁵Zn-labeled neutral proteinase from Streptomyces naraensis was prepared by the method described previously. The preparation was homogeneous by disc electrophoresis and contained 1 g-atom of zinc per mole of enzyme in calculation by radioactivity.

It was suggested that the protein-bound zinc of neutral proteinase was not essential for enzymatic activity. Thus, this zinc was an essential component for the higher order structure of the protein, and the removal of zinc treated with EDTA* inactivated the enzyme. The enzymatic activity was maintained in the presence of calcium ion.     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

嗜碱性芽孢杆菌PB92碱性蛋白酶分别在大肠杆菌和枯草芽孢杆菌中表达

从Bacillus alcalophillus PB92中扩增出碱性蛋白酶基因Mapr,Mapr分别插入到大肠杆菌载体pET-22b( )和枯草芽孢杆菌载体pWB980中构建成重组分泌型表达载体pET22b( )-Mapr、pWB980-Mapr。碱性蛋白酶基因分别在大肠杆菌宿主BL21和枯草芽孢杆菌DB104中得到表达。SDS-PAGE分析,重组蛋白酶的分子量为28kD。在大肠杆菌,所得酶活为231U/ml,而在枯草芽孢杆菌,其酶活为1563U/ml。大概是由于碱性蛋白酶在枯草芽孢杆菌折叠成熟机制与大肠杆菌的不同造成的。     

枯草芽孢杆菌(Bacillus subtilis)碱性脂肪酶基因在毕赤酵母中的表达

将来自枯草芽孢杆菌的碱性脂肪酶基因经密码子优化,全基因合成后克隆到pPICZαA载体,构建了pPICZαA-bsl分泌型重组质粒,该重组质粒经限制性内切酶PmeI线性化后使用LiCl法转化到毕赤酵母X-33,经过筛选获得分泌表达碱性脂肪酶的重组毕赤酵母X-33/pPICZαA-bsl。摇瓶发酵液上清酶活最高可达4.78 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为40-60℃,最适pH9.0,且具有高度耐碱的特性。该重组脂肪酶对旧新闻纸具备较明显的脱墨能力。