黑曲霉内切菊粉酶基因在毕赤酵母中的表达及应用研究

利用PCR方法从黑曲霉（Aspergillus niger）基因组DNA中扩增内切菊粉酶（endo I）全长基因（1551 bp）,经序列分析后将其连接到表达载体p PIC9K上,得到重组表达载体p PIC9K-endo I。重组质粒经Sac I线性化并电转化到毕赤酵母GS115,阳性转化子进行发酵产酶后考察其酶学性质。酶学性质研究表明,重组酶的最适p H和最适温度分别为5和60℃,重组酶在55℃下保温9 h后,重组菊粉内切酶仍残余83.2%的活性,表现出极高的热稳定性,Cu²⁺、Ag⁺对重组酶有明显的抑制作用。对内切菊粉酶水解菊粉的过程研究表明,重组内切菊粉酶能在8 h内将4%（w/v）菊粉水解为3⁶个聚合度的低聚果糖,水解11 h后,低聚果糖得率达到63.5%,本研究为进一步开发以菊粉为原料生产低聚果糖的应用奠定了基础。      

黑曲霉低聚葡萄糖氧化酶的分子克隆与生化特征

低聚葡萄糖氧化酶能够氧化低聚葡萄糖末端残基生成相应的低聚糖酸,在低聚糖酸的制备、食品、化工等方面具有潜在的应用价值。从黑曲霉基因组中发现1个疑似编码低聚葡萄糖氧化酶开放读框,共编码473个氨基酸序列,包含2个保守结构域(FAD结合域和黄素结构域)和4个活性位点(His80、Cys141、Asp377和Try428);在毕赤酵母中表达的重组酶蛋白大小为61 kDa,比酶活0.33 U/mg。重组酶的最适作用温度和pH值分别为75℃和9.0;在55～85℃和pH 5.0～9.0范围内孵育4 h,仍能保持70%以上的酶活;Mn²⁺和Fe³⁺分别对酶活具有最强的激活和抑制作用。该酶的K_m和V_max为0.48 mmol/L和2.22μmol/(L·min),对麦芽三糖、麦芽四糖和麦芽五糖具有相对较高酶活。     

黑曲霉XZ-3S木聚糖酶基因xynZF-2在毕赤酵母中的高效表达

将黑曲霉XZ-3S木聚糖酶基因xyn ZF-2(成熟肽碱基)插入表达载体p PIC9K中,SalⅠ线性化后电击转化毕赤酵母GS115,经MD平板、G418梯度和摇瓶复筛筛选出多克隆阳性转化子。选择28h的种子,每隔24h补加1.5%的甲醇,发酵96h后,比酶活可达13210U/mg;SDS-PAGE分析表明,该蛋白相对分子质量为23.0ku;重组酶最适作用温度为45℃,最适作用p H为5.0,在温度35~40℃、p H5~7条件下有良好的稳定性,Ca2+对酶的激活作用最明显。     

烟曲霉壳聚糖酶在毕赤酵母中的分泌表达

为了筛选高效表达壳聚糖酶的毕赤酵母菌株。重组质粒pPIC9K-CSN经SalⅠ线性化后,电击转化毕赤酵母GS115,表型筛选Mut+转化子,PCR鉴定后,用G418梯度浓度筛选多克隆子,用甲醇诱导摇瓶分泌表达。经PCR分析,质粒转到宿主菌GS115,在G418为4mg/mL时,筛选到了多克隆子,经SDS-PAGE分析表明分泌表达蛋白的相对分子量约为25kDa,酶活为14.59 U/mL。     

黑曲霉内切β-1,4-半乳聚糖酶AghA的分子克隆与特征解析

利用内切β-1,4-半乳聚糖酶(endo-β-1,4-galactanase,EC 3. 2. 1. 89)水解土豆浆制备低聚半乳糖,有望解决产品回收率低的问题。为此,该研究通过分子克隆技术,将黑曲霉β-1,4-半乳聚糖酶基因agh A在毕赤酵母中进行克隆表达,构建获得了重组菌GS115(p PIC-aghA)。摇瓶培养条件下,重组酶Agh A的酶活为130 U/m L,高于大多数已报道的半乳聚糖酶的酶活。酶学性质的研究表明,该酶的最适反应pH值和温度分别为4. 5和45℃,且在pH 4. 0～6. 0或30～50℃具有较好稳定性。大部分金属离子和EDTA对重组酶Agh A的酶活没有显著影响; Fe3+对其活性有强烈的抑制作用;而Hg2+则可使Agh A几乎完全失活。该酶对半乳聚糖的最大反应速率Vmax和米氏常数Km分别为400 mg/(m L·min)和0. 08 mg/m L。此外,重组酶Agh A还可以水解土豆浆,产生半乳二糖、半乳三糖和少量半乳四糖等低聚半乳糖。     

碱性木聚糖酶在毕赤酵母的表达及酶学性质研究

将教酒链霉菌L1105第10家族碱性木聚糖酶基因去除纤维素结合域(CBD)后将功能结构域基因xynAe SC克隆到毕赤酵母表达载体。经筛选得到重组工程菌GS1153-20,用甲醇诱导后产酶活力达到最高683±9U/m L。结果表明,重组木聚糖酶的最适p H为7.2,且在p H 6.2~8.6有较高的相对酶活;其最适温度为70℃,40~60℃时相对酶活力都在70%以上;重组酶Xyn Ae SC以燕麦木聚糖为底物时,酶活力最高,相对酶活力为桦木的259.7%±10.7%;对甘蔗渣木聚糖的酶活力最低,只有对桦木木聚糖酶活的16.0%±1.5%。研究表明,碱性木聚糖酶基因成功地在毕赤酵母中表达,且去除CBD域后,其酶学性质并未改变。     

黑曲霉中葡萄糖氧化酶基因的克隆及其在毕赤酵母中的表达

为了提高葡萄糖氧化酶的生产能力,提取和纯化了黑曲霉Aspergillus niger PCTC的基因组DNA,以此为模板进行PCR扩增获得葡萄糖氧化酶基因,经测序,所得基因全长1 772 bp,编码含有589个氨基酸的蛋白质。将目的基因和表达载体pPIC9K连接经电转化导入毕赤酵母GS115中,在甲醇诱导和α信号肽的转运下,将葡萄糖氧化酶分泌到胞外。经G418梯度抗性平板和显色平板的初筛以及摇床复筛,获得了一株产葡萄糖氧化酶活力较高的菌株,该株菌在30℃、180 r/min的培养条件下,经0.5%的甲醇诱导发酵4 d可获得0.342 U/mL的酶活。对该菌株进行了摇瓶产酶条件优化,其最佳发酵条件为:200 r/min、pH 5、接种体积分数50%、25℃下经1.5%甲醇诱导7 d,酶活达到25 U/mL。实验结果表明:葡萄糖氧化酶基因已经成功转入毕赤酵母,并获得相当的产量。     

黑曲霉葡萄糖氧化酶基因在毕赤酵母SMD1168中的表达

在毕赤酵母SMD1168中利用乙醇氧化酶AOX1强启动子表达黑曲霉葡萄糖氧化酶(Glucose oxidase,GOD)。提取黑曲霉Aspergillus niger PCTC的基因组DNA,以此为模板进行PCR扩增获得葡萄糖氧化酶基因,将目的基因插入到具有AOX1强启动子的表达载体p PICZαA上,经电转化导入毕赤酵母SMD1168中。经zeocin抗性平板初筛、摇床复筛以及SDS-PAGE蛋白质电泳的检测,获得了一株产葡萄糖氧化酶活力的菌株,该株菌在30℃、200 r/min的培养条件下,经体积分数1.0%的甲醇诱导发酵1 d可获得1.12 U/mL的酶活。对该菌株进行了摇瓶产酶条件优化,其最佳发酵条件为:在pH 5、30℃下经体积分数1.5%甲醇诱导7 d,酶活为32 U/mL。     

Constitutive expression,purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry

BACKGROUND: Pectin methylesterase (PME) catalyses the hydrolysis of the methyl ester of pectin, yielding free carboxyl groups and methanol. PME is widely used in the food, cosmetic and pharmaceutical industries. RESULTS: PME from Aspergillus niger was constitutively expressed to a high level in the yeast Pichia pastoris. The recombinant PME was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, giving an overall yield of 28.0%. It appeared as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular mass of about 45 kDa. Optimal activity of the enzyme occurred at a temperature of 50 °C and a pH of 4.7. The K_m, V_max and k_cat values of the enzyme with respect to pectin were 8.6 mmol L^?1 [ ], 1.376 mmol min^?1 mg^?1 and 8.26 × 10² s^?1 respectively. Cations such as K⁺, Mg²⁺, Ni²⁺, Mn²⁺ and Co²⁺ slightly inhibited its activity, whereas Na⁺ had no effect. CONCLUSION: PME from A. niger was constitutively expressed to a high level in P. pastoris without methanol induction. The recombinant PME was purified and characterised and shown to be a good candidate for potential application in the fruit juice industry. Copyright © 2012 Society of Chemical Industry     

黑曲霉脂肪酶tglE的基因克隆与生化特征解析

从黑曲霉基因组中克隆了一种新的脂肪酶基因tglE,并在毕赤酵母中成功表达。重组酶具有典型的脂肪酶活性,其最适pH为7. 0,最适温度为40℃;在30～60℃或pH 6. 5～9. 0该酶的酶活力保持在60%以上;在pH 6. 0～8. 0或20～30℃均较为稳定。Cu~(2+)、Mn~(2+)、Ca~(2+)、K~+、Mg~(2+)和Sn~(2+)对tgl E有明显的激活作用; Fe~(3+)、Zn~(2+)和EDTA对重组酶的酶活抑制作用明显,特别是EDTA可抑制重组酶90%的活。tgl E对橄榄油和C_4底物的作用最强,对棕果油和C16底物的作用最弱。以C_4为底物,tglE的Km值为14. 40 mmol/L,Vmax为46. 72mmol/(m L·h)。tgl E具有催化辛酸和乙醇生成辛酸乙酯的酯化活性。     

Cloning of levansucrase from Leuconostoc mesenteroides and its expression in Pichia pastoris

A gene (m1ft) encoding levansucrase from Leuconostoc mesenteroides has been previously cloned in Escherichia coli. Presently, m1ft was cloned and secretively expressed in Pichia pastoris. Methanol induction of recombinant M1FT resulted in the production of active levansucrase (PM1FT). PM1FT-5 was expressed as an active form, but the protein accumulated mainly inside the cells, representing about 5% of total cell proteins. M1FT was secreted into the culture supernatant with a maximum yield of 14,400 U/L using fed-batch fermentations. P. pastoris-derived M1FT displayed catalytic activities comparable to those of the E. coli-derived M1FT. PM1FT was glycosylated at its 2 potential N-glycosylation sites.     

黑曲霉果糖基水解酶的重组表达及酶学特征分析

目的:果糖基转移酶在新型果糖基衍生品的制备过程中具有重要作用,获得酶活高、性能优良的果糖基水解酶是关键。方法:利用MEGA 4.0以及Clustal X2等软件对黑曲霉的基因组进行分析,遴选了5个果糖基水解酶基因,接着将它们在毕赤酵母中进行了克隆表达,并研究了重组酶的酶学性质。结果:在摇瓶水平上,重组酶Fru1和Fru5的酶活分别为1360和1560 U/m L,远高于目前已报道的果糖基水解酶的酶活。重组酶Fru1的最适反应温度和p H分别为45℃和5.5,EDTA对它有微弱的促进作用,Fe²⁺、Na+、Co²⁺、Cu²⁺和Ca²⁺会轻微地抑制酶活,该酶对蔗糖既有水解作用,又有转苷活性,还可以水解菊粉中的二糖到五糖;重组酶Fru5的最适反应温度和p H分别为50℃和5.0,Li⁺、Na⁺和EDTA对其酶活有促进作用,但Fe²⁺则强烈抑制酶的活性,它还可以水解蔗糖以及菊粉中几乎所有的大分子糖。结论:本研究为果糖基水解酶的工业化生产及其在新型果糖基衍生品制备过程中的应用提供了可能途径。      

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

解淀粉芽杆菌中性植酸酶基因的克隆及其在毕赤酵母中的表达

采用PCR法从解淀粉芽孢杆菌BA11中克隆到一个中性植酸酶phyc基因,将该基因克隆到毕赤酵母表达载体pPIC9K上并电转化至宿主细胞GS115后进行诱导表达.SDS-PAGE试验表明,该重组中性植酸酶在毕赤酵母宿主细胞中实现了高效分泌性表达.植酸酶活性测定结果显示阳性克隆子在诱导72h酶活性达到最高值,活性为2330U/L.     

果糖基转移酶在酿酒酵母中异源表达及酶学性质分析

采用RT-PCR方法,获得编码黑曲霉YZ59果糖基转移酶的基因fwt,并在酿酒酵母中实现了异源表达。发酵48 h后,果糖基转移酶最高酶活力可达19.8 U/m L。纯化后,测定分析了果糖基转移酶的酶学性质。结果显示,果糖基转移酶FWT的最适温度为55℃,在低于50℃稳定。果糖基转移酶FWT的最适p H为5.5,稳定p H范围为4.0~9.0。果糖基转移酶FWT的Km和Vmax分别为169.5 g/L和0.7 g/(L·min)。Ni2+和Mg2+可显著激活果糖基转移酶FWT。     

Isolation and bioenergetic characterization of mitochondria from Pichia pastoris

Pichia pastoris is a methylotrophic yeast of high biotechnological interest. The bioenergetic properties of mitochondria from Pichia pastoris have not yet been determined. We report on a protocol for the isolation of the mitochondria in a state that shows good energy coupling. Analysis of Pichia pastoris growth and bioenergetic properties of the isolated mitochondria reveals that glycerol is the carbon source that yields the best results. Under our growth conditions, mitochondria oxidize external NADH but do not possess an alternative oxidase. Finally, Pichia pastoris mitochondria also lack the nucleotide-stimulated uncoupling pathway previously identified in Saccharomyces cerevisiae.     

Coprinopsis cinerea 漆酶基因的克隆及其在毕赤酵母中的表达

本文通过RT-PCR从Coprinopsis cinerea okayama7#130中克隆得到laccase1,应用SignalP3.0Server软件分析其氨基酸序列后设计不含信号肽序列的新引物扩增得到不含信号肽的漆酶基因1（lac1）,并构建重组酵母表达载体pPIC9K-lac1,电击转化毕赤酵母GS1115并用甲醇诱导表达,之后研究重组酶的酶学性质。克隆得到的laccase1全长为1593bp,编码530个氨基酸,其中信号肽包含18个氨基酸。SDS-PAGE显示重组蛋白大小约为65KDa。酶学性质研究表明,该酶最适反应温度为45℃,最适pH为4.3。重组漆酶在45℃保存3h后活性基本保持不变,在pH4.0～10.0的范围内稳定性较好。成功分泌表达的漆酶活力达到1.108U/mL。