Regulation mechanism of expression of the cloed pullulanase and development of its secretion system

普鲁兰聚糖酶是一种淀粉去支化酶，能在普鲁兰多糖和淀粉中水解(1-6)-α-糖苷键。普鲁兰酶用于阐明多糖的结构，并通过在有或没有其他淀粉酶的情况下使淀粉脱枝来产生有用的物质，如麦芽糖、直链淀粉和葡萄糖。这种酶是由革兰氏阴性细菌产气克雷伯氏菌产生的，并从细胞分泌到培养液中。我们最近克隆了普鲁兰酶基因(Pula)1。缺失分析和lac基因融合分析表明，pUL编码序列与调控基因完全位于4.2kb的染色体DNA片段内。结论：1.在麦芽糖诱导下，携带Plas-MID的产气克雷伯氏菌细胞胞外和胞内均产生过量的普鲁兰聚糖酶。我们测定了PUL基因的全部核苷酸序列。发现了一个3,288个碱基的独特开放阅读框。前体酶由1,096个氨基酸组成的res…。更多的IUDS，并含有疏水的N末端信号肽。前体蛋白和成熟蛋白的相对分子质量分别为119,334和117,258。核糖体结合位点位于ATG的上游8个碱基。在第230位(GGATGA)和260位(TCTAAT)检测到潜在的-35和-10启动子区域。E证实了Pula基因是由麦芽糖4诱导的麦芽糖控制的。检测了普鲁兰酶在大肠杆菌和产气性克雷伯菌中的细胞定位。在大肠杆菌中，大约50%的普鲁兰糖和46%的普鲁兰糖分别留在细胞膜和细胞质中。产气克雷伯氏菌在发酵液中释放出约20%的普鲁兰聚糖酶，在胞质组分和内外膜中各保留约20%的普鲁兰酶。氨基酸序列显示，在普鲁兰酶分子之前有一个19个氨基酸的信号肽，在潜在的切割位置有一个甘氨酸残基，前体在那里进行加工以产生成熟的普鲁兰酶。这个序列之后是半胱氨酸残基，它被棕榈酸酯修饰，很可能成为成熟蛋白质的氨基末端。亮氨酸-亮氨酸-甘氨酸-半胱氨酸序列是细菌脂蛋白的共同序列。较少

Pullulanase, a starch-debranching enzyme, hydrolyzes (1-6)- <alpha> -glucosidic linkages in pullulan and starch. Pullulanase is used to elucidate the structures of polysaccharides and to produce useful materials such as maltose, amylose, and glucose by debranching starch with and without other amylases. This enzyme is produced by the gram-negative bacterium, Klebsiella aerogenes, and secreted from the cells into the culture broth. We recently cloned the pullulanase gene (pulA).1. Deletion analysis and the lac gene fusion of the recombinant plasmid showed that the pul coding sequence, with the regulator gene, was located entirely within a 4.2-kb segment of the chromosomal DNA. The K. aerogenes cells carrying the plas-mid over-produced both extracellular and intracellular pullulanase when the cells were induced by maltose.2. We determined the entire nucleotide sequence of the pul gene. A unique open reading frame of 3,288 bp was found. The precursor enzyme consists of 1096 amino acid res … More idues and contains a hydrophobic N-terminal signal peptide. The molecular weight of precursor and mature proteins are estimated to be 119,334 and 117,258, respectively.3. A ribosome binding site is located 8 bases upstream of the ATG. Potential -35 and -10 promoter regions were detected at positions 230 (GGATGA)and 260 (TCTAAT). e confirmed that the pulA gene was controlled by malT, which is induced by maltose4. Cellular localization of pullulanase in E. coli and K. aerogenes carrying the plasmid were examined. In E.coli about 50 and 46% of the pullulanse remained in the membrane and cytoplasmic fractions, respectively. In K. aerogenes about 20% was released in the culture broth, and about 20% each of pullulanase remained in the cytoplasmic fraction and inner and outer membranes.5. The amino acid sequence showed that a 19-amino acid signal peptide preceded the pullulanase molecule with a glycine residue at the potential cleavage site, where processing of the precursor occured to yield the mature pullulanase. This se quence was followed by a cystein residue, which was modified by palmitate and probably becomes the amino terminus of the mature protein. A leu-leu-ser-gly-cys sequence is the consensus sequence for the bacterial lipoprotein. Less