Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis

A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.     

中性蛋白酶基因诱导型表达分泌载体的构建

利用PCR方法分别扩增出sacB基因的启动子-信号肽序列（sacR）和枯草芽孢杆菌中性蛋白酶的前肽-成熟肽序列,将两者连接后克隆入载体pHP13中,构建了含有中性蛋白酶基因的诱导型表达分泌载体pHP13SN,再将其转化入枯草杆菌DB104,获得基因工程菌DB104(pHP13SN)。中性蛋白酶基因在蔗糖的诱导和sacR的调控下实现了分泌表达,并获得了具有生物学活性的中性蛋白酶。     

磷脂酰丝氨酸合成酶基因的克隆及在枯草芽孢杆菌中的表达

应用 PCR技术从 Escherichia coli K12 Sgal- (ExPASy P23830) 中扩增到大小为 1350bp编码磷脂酰丝氨酸合成酶的 DNA片段,将其插入枯草芽孢杆菌诱导型表达载体 pBES,获得重组质粒 pBES-pss后转化 Bacillus subtilis DB104。经蔗糖诱导后,该磷脂酰丝氨酸合成酶在 Bacillus subtilis DB104 (pBES-pss)中获得胞外分泌表达,SDS-PAGE分析发现目的蛋白分子量约为 52kDa,酶联比色法检测酶活力为 1.50U/mL,提高了磷脂酰丝氨酸合成酶的表达产量,为工业化发酵生产磷脂酰丝氨酸合成酶奠定了良好的基础。     

alpha-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells

The alpha-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-epsilon-aminocaproyl-alpha-D-galactopyranosylamine as the ligand. From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9%. The cDNA coding for the alpha-galactosidase was cloned and sequenced. The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature alpha-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950. Seven cysteine residues were also found but no putative N-glycosylation sites were present. The observed homology between the deduced amino acid sequences of the mature enzyme and alpha-galactosidases from coffee (Coffea arabica), guar (Cyamopsis tetragonolooba), and Mortierella vinacea alpha-galactosidase II were over 73, 72, and 45%, respectively. The enzyme displayed maximum activity at 45 degrees C when p-nitrophenyl-alpha-D-galactopyranoside was used as substrate. The rice alpha-galactosidase and Mortierella vinacea alpha-galactosidase II acted on both the terminal alpha-galactosyl residue and the side-chain alpha-galactosyl residue of the galactomanno-oligosaccharides.     

Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected

The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.     

Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.     

双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。     

芽孢杆菌α-淀粉酶基因的克隆、表达和酶学性质分析

在仔猪结肠内容物中分离出一株能利用淀粉的芽孢杆菌Bacillussp.WS06,构建了全基因组DNA文库,从中筛选出α_淀粉酶基因amyF,分析测定了其核苷酸序列并进行了表达;其中amyF编码的蛋白有526个氨基酸、分子量为58.6kD;它与已报道的Bacillusmegaterium的α_淀粉酶序列有93%的同源性。经过氨基酸序列比较分析还发现,AmyF含有淀粉酶家族中4个高度保守的酶催化活性区。经多步纯化,重组酶的比活共提高了22.2倍,获得凝胶电泳均一的蛋白样品;经SDS_PAGE检测,AmyF酶分子量为57kD。该酶的最适反应温度为55℃～60℃,酶的最适反应pH为7.0,在温度不超过55℃时,酶活较稳定;AmyF能迅速降解淀粉生成麦芽寡糖,属于内切糖苷酶。     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.     

Preparation and Properties of a beta-d-Glucanase for the Specific Hydrolysis of beta-d-Glucans

A beta-d-glucanase highly specific for glucans containing a linkage sequence ... Glc 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc ... has been isolated from several commercial preparations of Bacillus subtilis alpha-amylase including one purified by repeated crystallization. The beta-d-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the alpha-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing sugars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity at pH 6.5, a V(max) of 13.9 mug glucose equivalent released/mug protein.hour, and an apparent Km of 3.4 mg of lichenan per ml. Potential application of this enzyme for the structural characterization of plant cell wall glucans is discussed.     

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.     

Specific binding of lactoferrin to Aeromonas hydrophila

The subunit S1 of pertussis toxin (PT) was purified as the recombinant product BacS1 from the culture supernatant of a Bacillus subtilis strain containing a secretion vector with a DNA fragment coding for the mature subunit S1 inserted downstream of the signal sequence of the alpha-amylase gene. The method of purification was successive ion exchange and adsorption chromatography. BacS1 occurred in two forms (28 and 20 kDa) of which the truncated 20-kDa peptide was the main one in the supernatant. The truncated BacS1 was purified and shown to have the same NH2-terminus as the full-size (28 kDa) BacS1. It was also enzymatically active indicating correct conformation. The truncated BacS1 was also shown to elicit neutralizing and protective antibodies when injected into mice or rabbits.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.