Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.     

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.     

Rapid access to genes of biotechnologically useful enzymes by partial genome sequencing: the thermoalkaliphile Anaerobranca gottschalkii

Anaerobranca gottschalkii strain LBS3 T is an extremophile living at high temperature (up to 65 degrees C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two alpha-amylases (AmyA and AmyB), and an alpha-1,4-glucan-branching enzyme.     

Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiella aerogenes

We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.     

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.     

Collagen-like sequences stabilize homotrimers of a bacterial hydrolase.

Pullulanase from Klebsiella pneumoniae strain FG9 has an unusual N-terminal amino acid sequence that includes six repeats of the tripeptide Gly-X-Pro. This type of sequence is characteristic of animal collagens and collagen-like proteins which form triple helical structures. We have investigated the molecular organization of this bacterial pullulanase isolated from the cell surface of Escherichia coli cells that carry the cloned FG9 pulA (pullulanase encoding) gene. Non-denaturing polyacrylamide gel analysis shows that pullulanase exists as higher order, apparently homogeneous, structures. We have used highly purified bacterial collagenase to probe the role of the collagen-like region and we demonstrate that this feature is essential for non-covalent association of pullulanase homotrimers. In addition we show collagenase-specific release of cell-bound pullulanase.     

Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.     

Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5-7 and 80°C and had a half-life of 88 min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity.     

Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228

The gene that coded for a cellular pullulanase of type I (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82 degrees C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15-21% and 43% identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively. Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (beta/alpha)8-barrel domain, one domain made of the region protruding from the barrel between the third beta-strand and the third alpha-helix, and one beta-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the beta-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain.     

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium,Caldicellulosiruptor saccharolyticus

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95 732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5′-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.     

丝状支原体丝状亚种SC型脂蛋白Q N末端基因的表达、纯化与抗原性分析

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。     

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。     

Expression and biochemical characterization of a novel type I pullulanase from <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Objectives

To identify novel pullulanases from microorganisms and to investigate their biochemical characterizations.

Results

A novel pullulanase gene (BmPul) from Bacillus megaterium WW1210 was cloned and heterologously expressed in Escherichia coli. The gene has an ORF of 2814 bp encoding 937 amino acids. The recombinant pullulanase (BmPul) was purified to homogeneity and biochemically characterized. BmPul has an MW of approx. 112 kDa as indicated by SDS-PAGE. Optimum conditions were at 55 °C and pH 6.5. The enzyme was stable below 40 °C and from pH 6.5?8.5. The Km values of BmPul towards pullulan and amylopectin were 3.3 and 3.6 mg/ml, respectively. BmPul hydrolyzed pullulan to yield mainly maltotriose, indicating that it should be a type I pullulanase.

Conclusions

A novel type I pullulanase from Bacillus megaterium was identified, heterologously expressed and biochemically characterized. Its properties makes this enzyme as a good candidate for the food industry.

     

Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

[目的]鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因;研究其在大肠杆菌系统的克隆与表达;分析该酶与其同源酶的活性中心氨基酸序列.[方法]从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus;CCTCC M203062)发酵液中,分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase),测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物,扩增得到pro-MTGase 基因,将该基因插入到表达载体pET-20b( )信号肽pelB下游,构建分泌型表达载体pET/pro-MTG,并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS.[结果]获得了pro-MTGase的完整基因序列,多重碱基序列比对表明其与S.platensis和S.caniferus的pro-MTGase基因同源性高达92%.利用Rosetta(DE3)pLysS通过降温至24℃诱导策略,获得部分胞外表达的酶原.SDS-PAGE显示,胞外表达重组蛋白的分子量约为44kDa,与吸水链霉菌表达的天然酶原相符.诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL.[结论]该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道,也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达.     

尿酸氧化酶基因的克隆、表达及其产物的应用

克隆了产朊假丝酵母(Candida utilis)AS2-117尿酸氧化酶(Urate Oxidase,Uricase,EC1.7.3.3)的基因。将此基因插入原核表达质粒pET21a后转化大肠杆菌BL21(DE3),获得高表达的重组转化子菌株。经IPTG诱导,重组尿酸酶基因表达量可达菌体可溶性蛋白的40%。重组尿酸氧化酶为有酶活性的可溶蛋白。Western印迹分析证实表达产物有免疫学活性。经DEAE DE52纤维素离子交换柱层析纯化,目的蛋白纯度可达95%。重组蛋白和天然蛋白的理化特征比较证明重组蛋白的热稳定性有较大提高。酶盒配制和临床应用实验表明重组蛋白可代替天然蛋白进行临床血清尿酸的分析。     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

人胶源神经营养因子基因的克隆及在大肠杆菌中的表达

胶源神经营养因子（Ｇｌｉａｌｃｅｌｌｉｎｅｄｅｒｉｖｅｄｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ＧＤＮＦ)是大鼠B49细胞系中分离纯化得到的一种蛋白质^[1],由于其对多巴胺神经元的专一性的神经营养作用而被发现。GDNF成熟蛋白由134个氨基酸组成,具有两个N-糖基化位点。它属于TGF-β基因家族但与该家族其它成员的氨基酸序列同源性仅为20%,可能是一个新的亚家族。最近研究表明,它对发育中的运动神经元也有很强的神经营养作用^[1]。对啮齿类^[3,4],灵长类的弥猴^[5]的活体试验表明,胶源神经营养因子是一种治疗神经退化发夹知病如帕金森氏症、肌萎缩性脊髓索硬化症等的非常有效的潜在药物。由于GDNF在体内含量极低而且公在发育早期表达,因而只有通过基因工程方法才能获得大量的GDNF。本文报道采用PCR方法从中国入基因组DNA 中扩增出编码GDNF的基因,并实现在大肠杆菌中的高效表达。这为进一步研究GDNF的结构和生物学功能打下了坚实的基础。