枯草芽孢杆菌BJ3-2赖氨酸脱羧酶基因yaaO的克隆与序列分析

根据GenBank中的枯草芽孢杆菌（Bacillus subtilis） 168菌株假定的赖氨酸脱羧酶基因（LDC）序列设计同源引物,提取枯草芽孢杆菌BJ3-2基因组DNA进行PCR扩增,并克隆至pGEM-T载体后测序.序列分析结果表明：Bacillus subtilis BJ3-2 yaaO基因开放阅读框（ORE）长为1 443bp,获得基因登录号为KJ561349,BLAST比对与已报道枯草芽孢杆菌的yaaO基因核苷酸序列及氨基酸序列同源性均高达90％以上,对赖氨酸脱羧酶氨基酸序列构建系统进化树分析,发现Bacillus subtilis BJ3-2的LDC与B.subtilis JS亲缘关系最近,SWISS-MODEL预测该蛋白的3D结构与鸟氨酸/赖氨酸/精氨酸脱羧酶蛋白相似性为39.22％,属于典型的Ⅲ型磷酸吡哆醛（PLP）依赖型鸟氨酸/赖氨酸/精氨酸脱羧酶家族成员.yaaO基因序列分析及氨基酸保守结构域的分析,为有效控制水豆豉产品中尸胺含量过高的研究提供了理论基础.     

枯草芽孢杆菌精氨酸脱羧酶基因speA的表达与蛋白纯化

根据枯草芽孢杆菌（Bacillus subtilis）BJ3-2的精氨酸脱羧酶（ADC）的编码基因speA序列设计特异性酶切引物,克隆基因speA序列。测序结果显示,基因speA全长为1 473 bp,编码490个氨基酸,分子质量为58 ku。基因speA克隆至原核表达载体,获得重组菌pET28a-speA/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示,1.0 mmol/L的异丙基β-D-硫代半乳糖苷（IPTG）28 ℃诱导4 h,上清液和菌体均能表达出ADC蛋白,上清液经纯化、透析、冷冻干燥可获得纯度97%的ADC酶,酶联免疫吸附检测（ELISA）ADC酶活为16 780 U/mg。为speA基因的表达、纯化及酶学性质研究奠定了理论基础。     

枯草芽孢杆菌BJ3-2 S-腺苷甲硫氨酸脱羧酶基因speD的克隆与序列分析

S-腺苷甲硫氨酸脱羧酶（SAMDC）是多胺合成过程中的限速酶。根据GenBank中枯草芽孢杆菌168菌株的S-腺苷甲硫氨酸脱羧酶基因（speD）序列设计同源引物,PCR扩增Bacillus subtilis BJ3-2gDNA,并克隆至pGEM-T载体后测序。序列分析结果表明：B. subtilis BJ3-2 speD基因ORF为381 bp,可编码126个氨基酸,分子质量为13.87 ku,基因登录号为KJ561347,Blast比对,SAMDC基因和蛋白序列的同源性都达到90%以上。B. subtilis BJ3-2的SAMDC具有丙酮酸依赖型脱羧酶的酶原剪切结构域[GVSGVVIISESHLTIH]和保守结构域[TCG],属于典型的I类B型SAMDC家族。SAMDC在多胺生物合成过程中扮演重要的角色,是多胺合成途径中的关键酶。研究为控制发酵豆豉中生物胺含量奠定了基础。     

枯草芽孢杆菌胺氧化酶基因的克隆与表达

以细菌型豆豉工业发酵菌种枯草芽孢杆菌（Bacillus subtilis）BJ3-2为材料,根据NCBI中菌株B. subtilis 168的胺氧化酶（AMO）基因序列设计同源引物,克隆获得菌株B. subtilis BJ3-2的胺氧化酶基因YobN序列。测序结果显示,YobN基因开放阅读框（ORF）长为1 437 bp,编码478个氨基酸,分子质量为53.79 ku,与菌株B. subtilis 168同源性达98%。目的基因克隆至原核表达载体,获得重组菌pET28a-YobN/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示,浓度为1.0 mmol/L的异丙基硫代半乳糖苷（IPTG）22 ℃诱导5 h,表达量最高达1.30 mg/mL。该研究为AMO活性的研究奠定了基础,对豆类发酵食品中生物胺的控制具有重要意义。     

高活性谷氨酰胺酶基因glsA2 在枯草芽孢杆菌BJ3-2染色体上的定点整合

以枯草芽孢杆菌BJ3-2 的glsA1 基因为同源序列,通过构建单交换整合载体,将高活性谷氨酰胺酶基因glsA2 定点整合入BJ3-2 菌株染色体中,获得能够稳定遗传的重组菌株BJ3-2A2。经检测重组菌株谷氨酰胺酶活力为BJ3-2 菌株的3.36 倍。利用重组菌株BJ3-2A2 发酵豆豉,全氨基酸检测显示,重组菌株发酵的豆豉谷氨酸含量比出发菌株高12.8%。说明枯草芽孢杆菌BJ3-2 可以转化且为RecE+ 菌株,glsA2 基因在BJ3-2 菌株染色体上可实现较高活性表达,并提高发酵豆豉的鲜味。     

高活性谷氨酰胺酶基因glsA_2在枯草芽孢杆菌BJ3-2染色体上的定点整合

以枯草芽孢杆菌BJ3-2的glsA1基因为同源序列,通过构建单交换整合载体,将高活性谷氨酰胺酶基因glsA2定点整合入BJ3-2菌株染色体中,获得能够稳定遗传的重组菌株BJ3-2A2。经检测重组菌株谷氨酰胺酶活力为BJ3-2菌株的3.36倍。利用重组菌株BJ3-2A2发酵豆豉,全氨基酸检测显示,重组菌株发酵的豆豉谷氨酸含量比出发菌株高12.8%。说明枯草芽孢杆菌BJ3-2可以转化且为RecE+菌株,glsA2基因在BJ3-2菌株染色体上可实现较高活性表达,并提高发酵豆豉的鲜味。     

枯草芽孢杆菌基因的安全改良研究

为了构建能稳定表达外源基因且无抗性标记基因的枯草芽孢杆菌(Bacillus subtilis)工程菌。采用聚合酶链式反应(PCR)技术,分两段扩增定位于B.subtilis BJ3-2染色体上的谷氨酰胺酶基因(glsA)上下游基因片段作为同源臂。将同源臂及卡那霉素基因(Kan)克隆入温敏型载体pKSV7,构建了双交换载体pKUKD。并利用pKUKD质粒将kan基因定点整合入BJ3-2染色体上,通过Kan抗性筛选获得glsA敲除菌株BJ-kan。经检测BJ3-2菌株谷氨酰胺酶活力比重组菌株高3.2倍。结果显示:BJ3-2染色体上的基因可通过同源重组方式进行敲除与置换,为今后BJ3-2菌株以及野生型B.subtilis基因敲除与置换提供了更加便捷的方法。     

一株芽孢表面稳定展示海藻糖合酶工程菌的构建

以枯草芽孢杆菌(Bacillus subtilis)168-Tres基因组为模板,PCR扩增得到同源臂基因sleB1和cwlJ1,重叠PCR连接sleB1与卡那霉素抗性(kmr )基因,电转获得B. subtilis 168-TresΔsleB菌株;连接cwlJ1与博来霉素抗性(zeor)基因,电转获得B. subtilis 168-TresΔsleBΔcwlJ菌株。结果表明,经kmr、zeor抗性筛选及PCR鉴定,成功获得sleB、cwlJ基因双缺失菌株B. subtilis 168-TresΔsleBΔcwlJ;发酵结果显示,B. subtilis 168-TresΔsleBΔcwlJ与出发菌株的芽孢形成率一致,约为88%;在LB固体培养基和麦芽糖转化生成海藻糖体系中B. subtilis 168-Tres的芽孢萌发数为4.8×108 CFU/mL,B. subtilis 168-TresΔsleBΔcwlJ芽孢未萌发;在麦芽糖转化生成海藻糖体系中,重组菌海藻糖合酶酶活为10.42 U,比原始菌提高了78.7%。敲除sleB、cwlJ基因后,不影响枯草芽孢杆菌生成芽孢的量,但能有效控制芽孢在上述转化体系中的萌发,使芽孢表面稳定展示海藻糖合酶,提高了芽孢的利用率。     

利用枯草芽孢衣壳蛋白表面展示β-半乳糖苷酶

分别将枯草芽孢杆菌(Bacillus subtilis 168)芽孢衣壳蛋白CotB、CotC、CotG和CotX的启动子和编码序列与来自嗜热脂肪芽孢杆菌(Bacillus stearothermophilus IAM11001)的β-半乳糖苷酶基因bgaB进行重组,构建融合表达cotB-bgaB、cotC-bgaB、cotG-bgaB和cotX-bgaB的整合型重组质粒。将4种重组质粒分别转入枯草芽孢杆菌Bacillus subtilis 168(trp-),获得了能在芽孢表面展示的重组菌株PB701、PB702、PB703和PB704。经Western blot检测,4种重组菌株均表达了预期分子量的融合蛋白,初步表明β-半乳糖苷酶被锚定在重组菌株的芽孢表面。以oNPG为底物测定4种重组菌株芽孢表面展示β-半乳糖苷酶的水解能力,得到的酶活分别为0.14、0.06、0.22和0.20 U/mL。     

土豆烧牛肉方便菜肴胀袋微生物分离鉴定及产气特性研究

为解决土豆烧牛肉方便菜肴产品的胀袋问题,对胀袋微生物进行分离、纯化、鉴定和产气特性研究。分离、纯化得到7株优势微生物。结合形态学观察、16SrRNA序列分析及基质辅助激光解析/电离飞行时间质谱(MALDI-TOF MS)技术鉴定分离微生物,结果显示2株为枯草芽孢杆菌(Bacillus subtilis), 2株为表皮葡萄球菌(Staphylococcusepidermidis), 2株为地衣芽孢杆菌(Bacillus licheniformis),1株为凝结芽孢杆菌(Bacillus coagulans)。将分离菌株接种到无菌土豆烧牛肉样品中,发现枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniformis)有明显产气现象,是引起产品胀袋的主要原因。     

Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods

The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.     

纳豆激酶基因工程菌的构建及酶活力分析

纳豆激酶由纳豆芽孢杆菌(Bacillus subtilis natto)的aprN基因编码,在体内外具有很强的溶解纤维蛋白活性。利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增B.subtilis natto的aprN基因,并依据B.subtilis的密码子偏好性优化了起始30个氨基酸的密码子,构建了重组表达质粒pHT01-aprN。经限制性酶酶切、PCR扩增和测序验证了其编码的正确性。通过电击法将含有强启动子的pHT01-aprN导入B.subtilis,利用氯霉素抗性筛选获得B.s 168/pHT01-aprN工程菌。经IPTG诱导表达,摇瓶发酵培养最高酶活力为(289.00±3.42)U/mL,是野生菌的3.9倍,酶活力表达稳定性良好。     

Difference in transcription levels of cap genes for gamma-polyglutamic acid production between Bacillus subtilis IFO 16449 and Marburg 168

In a strain carrying capB-lacZ fusion of Bacillus subtilis IFO16449, which produces a large amount of gamma-polyglutamic acid (PGA), beta-galactosidase activity was enhanced by about five times with the addition of L-glutamic acid. This increase was also confirmed by Northern blot analysis. On the other hand, the activity was not detected in a strain carrying capB-lacZ fusion of B. subtilis Marburg 168. However, when the cap genes (capBCA and ywtC) were fused to the IPTG-inducible spac promoter, B. subtilis Marburg 168 produced PGA. These results suggest that the inability of B. subtilis Marburg 168 to produce PGA is due to defective expression of the cap genes.