蜡样芽胞杆菌GXBC-3三个普鲁兰酶基因的表达及其酶学特性

探索获得优良的新型普鲁兰酶基因,丰富普鲁兰酶理论,对实现普鲁兰酶国产化具有重要意义。分析GenBank数据库中蜡样芽胞杆菌假定Ⅰ型、Ⅱ型普鲁兰酶基因序列,从实验室保藏的蜡样芽胞杆菌Bacilluscereus GXBC-3中克隆得到3个普鲁兰酶基因pulA、pulB、pulC,并分别导入大肠杆菌进行胞内诱导表达。纯化重组酶酶学性质研究表明重组酶PulA能水解α-l,6-和α-l,4-糖苷键,为Ⅱ型普鲁兰酶,以普鲁兰糖为底物时,最适反应温度及pH分别为40℃和6.5,比活力为32.89 U/mg;以可溶性淀粉为底物时,最适反应温度及pH分别为50℃和7.0,比活力为25.71 U/mg。重组酶PulB和PulC二者均只能水解α-l,6-糖苷键,为I型普鲁兰酶,以普鲁兰糖为底物时,其最适反应温度及pH分别为45℃、7.0和45℃、6.5,比活力分别为228.54 U/mg和229.65 U/mg。     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.     

凝结芽孢杆菌耐热β­N­乙酰氨基葡萄糖苷酶在大肠杆菌的分泌表达及其在制备GlcNAc中的应用

β-N-乙酰氨基葡萄糖苷酶体,可作用于几丁质或壳聚糖等天然底物,从末端水解产生N-乙酰-β-D氨基葡萄糖 (GlcNAc) 单体,其在医药和农业领域有较广泛的用途。文中克隆了耐热菌凝结芽孢杆菌Bacillus coagulans DMS1的β-N-乙酰氨基葡萄糖苷酶基因 (BcNagZ),并成功在大肠杆菌Escherichia coli BL21(DE3) 进行了分泌表达,蛋白表达量达到0.76 mg/mL。纯化后的BcNagZ分子量为61.3 kDa,测得的比活力为5.918 U/mg;进一步对该酶进行表征,结果显示酶的最适反应pH为5.5,最适反应温度为75 ℃,在65 ℃处理30 min后还有85%的残余酶活力,表明该酶具有良好的热稳定性。该酶的米氏常数Km为0.23 mmol/L,Vmax为0.043 1 mmol/(L·min)。重组BcNagZ可以水解胶体几丁质得到微量的GlcNAc,可以将二糖水解为单糖;偶联已报道的外切几丁质酶AMcase,可以有效地将胶体几丁质水解为GlcNAc,得率达到86.93%。     

Secreted expression of the pullulanase in Bacillus amyloliquefaciens BF7658

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP.将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游,获得重组质粒pUC980-BdP,重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株.摇瓶发酵实验表明,重组转化子发酵液有明显普鲁兰酶酶活,约48 h酶活达到最高水平,为2.8 ASPU/mL.酶学性质分析表明,重组酶最适作用温度约为60℃,最适反应pH为5.0,60℃保温3h仍保存50％的活性.重组酶性质适合淀粉糖化工艺的要求.     

青霉菌聚半乳糖醛酸酶基因在毕赤酵母中的表达及酶学性质研究

筛选得到一株能分解果胶的青霉菌(Penicillium sp.),使用简并引物PCR和TAIL-PCR方法从该菌中克隆了一个聚半乳糖醛酸酶基因pgp1.pgp1基因全长1 225 bp,包含2个内含子,其cDNA全长1 104 bp,编码367个氨基酸和一个终止密码子,前18个氨基酸为信号肽序列.将pgp1基因连接pPIC9载体,在巴斯德毕赤酵母表达系统中进行了异源表达.在3L发酵罐水平,培养基中聚半乳糖醛酸酶活力达到700 U/mL.酶学性质测定表明,重组酶蛋白PGP1的最适pH为5.0,在pH4.0 -6.0下处理1h后,剩余酶活力超过90％;最适温度为38℃,以聚半乳糖醛酸为底物,PGP1的Km=(1.172±0.169)mg/mL,Vmax=(0.061±0.002) mg/min/mL.     

长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控

[目的]从长野芽胞杆菌(Bacillus naganoensis)JNB-1中克隆出普鲁兰酶基因并在大肠杆菌系统中表达,通过优化诱导条件和使用化学添加剂提高了胞外酶活.[方法]采用PCR方法,从B.naganoensis基因组中扩增出普鲁兰酶基因pul,构建重组菌E,coli BL21/pET-20b-pul.通过优化,确定优化后的IPTG诱导条件以及甘氨酸、Na+的最佳添加参数.[结果]普鲁兰酶在大肠杆菌中得到有效表达,其相对分子质量为ll9kDa.在优化后的诱导条件(诱导温度20℃,IPTG终浓度0.4 mmol/L,在菌体OD600至1.2时诱导)下,普鲁兰酶的总酶活达到10.8 U/mL.添加甘氨酸和Na+均能有效促进普鲁兰酶的分泌.在诱导时添加终浓度0.08 mol/L的甘氨酸和0.2 mol/L Na+,胞外酶活提高至8.1 U/mL,是不加任何添加剂的10.3倍.[结论]该重组菌的构建为普鲁兰酶制剂的工业生产提供了有价值的菌株,对化学添加剂促进分泌的研究也为重组酶的高水平胞外生产提供了有效的方法.     

长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的表达及酶学性质研究

根据NCBI上报道的基因序列设计引物,以长野芽孢杆菌(Bacillus naganoensis)ATCC53909的染色体DNA为模板,PCR扩增普鲁兰酶编码基因pulB。将此基因与表达载体pWB980连接构建重组质粒pWB-pulB,并转化枯草芽孢杆菌WB600。SDS-PAGE结果显示,在100 kD处有特异性条带,经测定重组转化子粗酶液酶活力达10.94 U/mL。酶学性质分析表明,其最适反应温度为60℃,最适反应pH为5.0,且在温度30-60℃及pH4.0-6.0范围内稳定,适合淀粉加工行业的应用。     

碱性α-淀粉酶基因在巨大芽孢杆菌中的表达及酶学性质研究

将已克隆的碱性α-淀粉酶基因信号肽编码序列去除,用PCR的方法加入酶切位点,然后与表达载体pHIS1525连接转化大肠杆菌DH5α,筛选出阳性转化子DH5α-pHIS1525-JH,并提取质粒进一步转化巨大芽孢杆菌YYBm1原生质体,获得基因工程菌YYBm1 -pHIS1525-JH.SDS-PAGE分析表明该基因在巨大芽孢杆菌中得到了有效表达.酶学性质研究表明,该酶的最适温度与pH值分别为60℃与pH8.5,在pH7.0 - 10.5之间具有较好的稳定性,Km值为1.94 mg/mL,酶活力可达2516.5 U.     

α-葡萄糖苷酶菌株的选育及发酵条件的研究

从2株芽胞杆菌中通过筛选获得了一株产α-葡萄糖苷酶活力较高的嗜热脂肪芽孢杆菌U2,以嗜热芽孢杆菌U2为菌种,优化发酵培养基后,在温度45℃、初始pH6.8、转速200r/min和10%接种量条件下,发酵20h,菌株U2产酶水平可达到2.62U/mL,比出发菌提高了4倍。     

普鲁兰酶基因在解淀粉芽孢杆菌BF7658菌株中的分泌表达

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP。将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游,获得重组质粒pUC980-BdP,重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株。摇瓶发酵实验表明,重组转化子发酵液有明显普鲁兰酶酶活,约48h酶活达到最高水平,为2．8ASPU／mL。酶学性质分析表明,重组酶最适作用温度约为60℃,最适反应pH为5．0,60℃保温3h仍保存50％的活性。重组酶性质适合淀粉糖化工艺的要求。     

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

海栖热袍杆菌来源的极耐热碱性果胶裂解酶的表达、纯化及定性

将来源于极端嗜热菌属海栖热袍杆菌Thermotoga maritima MSB8的编码碱性果胶裂解酶的结构基因pelA与新型热激质粒pHsh连接, 得到重组质粒pHsh-pelA, 运用mRNA二级结构预测软件对pHsh-pelA的翻译起始区的二级结构进行优化, 得到了具有最佳mRNA二级结构及自由能的质粒pHsh-pelC。将重组质粒pHsh-pelC转入大肠杆菌JM109(DE3)进行表达, 得到了一种极耐热性碱性果胶裂解酶(PelC)。对重组酶的酶学性质研究发现, 该酶的最适反应温度为90oC, 最适反应pH为8.5, 在pH 8.2~9.8之间酶活力稳定, 95oC酶活半衰期为2 h, 并且该酶依赖Ca2+作为活性离子。在工业生产常用温度60oC下, 该酶能够长时间保持稳定, 并具有较高的酶活力。以多聚半乳糖醛酸(PGA)为底物时, 其动力学参数Km值为0.11 mmol/L, Vmax值为327 U/mg。SDS-PAGE结果显示该重组酶的分子量为43 kD, 与理论值相符。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点, 且重组酶热稳定性非常好, 这对该酶的大规模发酵应用具有重要意义。     

Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85.

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pI of 4.8, and temperature and pH optima of 39 degrees C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the Vmax was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a Vmax of 10.4 U/mg. It was a basic protein with a pI of 9.4, a temperature optimum of 39 degrees C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.     

一株高乙醇耐受的嗜热细菌Anoxybacillus sp. WP06的性质研究

厌氧芽孢杆菌属(Anoxybacillus)的菌株WP06是一株兼性厌氧的嗜热细菌, 能利用木糖、阿拉伯糖和葡萄糖等产生乙醇。不像绝大多数嗜热细菌, WP06菌株在高温下表现出极高的乙醇耐受力, 60oC时在8%的乙醇胁迫下才出现生长抑制现象, 15%的乙醇胁迫下仍能生长, 是目前已知的乙醇耐受力最高的嗜热细菌。WP06菌株突破了人们对高温下细菌耐受乙醇浓度的极限认识, 是研究高温下乙醇耐受机制的良好出发菌株。     

一株高乙醇耐受的嗜热细菌Anoxybacillus sp. WP06的性质研究

厌氧芽孢杆菌属(Anoxybacillus)的菌株WP06是一株兼性厌氧的嗜热细菌, 能利用木糖、阿拉伯糖和葡萄糖等产生乙醇。不像绝大多数嗜热细菌, WP06菌株在高温下表现出极高的乙醇耐受力, 60oC时在8%的乙醇胁迫下才出现生长抑制现象, 15%的乙醇胁迫下仍能生长, 是目前已知的乙醇耐受力最高的嗜热细菌。WP06菌株突破了人们对高温下细菌耐受乙醇浓度的极限认识, 是研究高温下乙醇耐受机制的良好出发菌株。     

Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate

An alkalophilic bacterial isolate identified as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 degrees C. The Km (for casein) was 11 mg/ml and Vmax was 380-microg tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 degrees C with a pH and temperature optimum at 8.4 and 40 degrees C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production.     

Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.     

Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.