Cloning of a foreign gene coding for alpha-amylase in Bacillus subtilis.

Foreign DNA has been introduced into the genome of bacteriophage Ø3T, producing a specialized transducing bacteriophage containing the genetic information encoding α-amylase from $\text{Bacillus}\text{amyloliquefaciens}$H. Genetic and physical studies demonstrated that the gene(s) is inserted into the bacteriophage genome. These bacteriophage carrying the gene(s) encoding α-amylase lysogenized and replicated in $\text{Bacillus}\text{subtilis}$ with normal efficiency. In these lysogens, the gene(s) encoding α-amylase appears to map near the bacteriophage attachment site rather than the chromosomal $\text{amy}$E locus. This method of construction of specialized bacteriophage should be applicable to the cloning of other genes for which no primary selection exists.     

Cloning and expression of a serine proteinase gene from Thermoactinomyces sp. in Bacillus subtilis cells

A gene coding for thermostable serine protease from Thermoactinomyces sp. K50 is cloned and expressed in Bacillus subtilis cells. Restriction map of cloned DNA fragment is determined. Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp. K50. Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.     

Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) at 1.6 A resolution and alpha-amylase 2 (TVAII) at 2.3 A resolution

The X-ray crystal structures of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) and alpha-amylase 2 (TVAII) have been determined at 1.6 A and 2.3 A resolution, respectively. The structures of TVAI and TVAII have been refined, R-factor of 0.182 (R(free)=0.206) and 0.179 (0.224), respectively, with good chemical geometries. Both TVAI and TVAII have four domains, N, A, B and C, and all very similar in structure. However, there are some differences in the structures between them. Domain N of TVAI interacts strongly with domains A and B, giving a spherical shape structure to the enzyme, while domain N of TVAII is isolated from the other domains, which leads to the formation of a dimer. TVAI has three bound Ca ions, whereas TVAII has only one. TVAI has eight extra loops compared to TVAII, while TVAII has two extra loops compared to TVAI. TVAI can hydrolyze substrates more efficiently than TVAII with a high molecular mass such as starch, while TVAII is much more active against cyclodextrins than TVAI and other alpha-amylases. A structural comparison of the active sites has clearly revealed this difference in substrate specificity.     

Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene.

Gene fusions of DNA sequences encoding protein A from Staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from Bacillus amyloliquefaciens (amyEBamP) directed the synthesis and efficient secretion of protein A in Bacillus subtilis. The fusions were established on multicopy pUB110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in B. subtilis. Some of the resulting B. subtilis strains secreted protein A at levels in excess of 1 g/liter, demonstrating that a foreign protein encoded by an engineered gene can be secreted by B. subtilis at levels comparable to endogenous exoproteins.     

枯芽孢杆菌α—淀粉酶基在在大肠杆菌中的表达及其产物的分泌

The E. coli which carrying the alpha-amylase gene fragment cloned from B. subtilis secreted the gene products into the medium. The reason is the exogenous gene fragment act on the cell wall of E. coli by some way, gives rise to the change of its structure. It leads up to the alpha-amylase and some periplasm proteins passing through the cell wall into the medium. It also causes the change of host colonial morphology. The secrete process are non-specific.     

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.     

Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

The gene coding for alpha-amylase from Bacillus amyloliquefaciens was isolated by direct shotgun cloning using B. subtilis as a host. The genome of B. amyloliquefaciens was partially digested with the restriction endonuclease MboI and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. One of the transformants producing high amounts of alpha-amylase was characterized further. The alpha-amylase gene was shown to be present in a 2.3-kb insert. The alpha-amylase production of the transformed B. subtilis could be prevented by inserting lambda DNA fragments into unique sites of EcoRI, HindIII and KpnI in the insert. Foreign DNA inserted into a unique ClaI site failed to affect the alpha-amylase production. The amount of alpha-amylase activity produced by this transformed B. subtilis was about 2500-fold higher than that for the wild-type B. subtilis Marburg strain, and about 5 times higher than the activity produced by the donor B. amyloliquefaciens strain. Virtually all of the alpha-amylase was secreted into the culture medium. The secreted alpha-amylase was shown to be indistinguishable from that of B. amyloliquefaciens as based on immunological and biochemical criteria.     

Alpha-amylase genes (amyR2 and amyE+) from an alpha-amylase-hyperproducing Bacillus subtilis strain: molecular cloning and nucleotide sequences.

amyR2, amyE+, and aroI+ alleles from an alpha-amylase-hyperproducing strain, Bacillus subtilis NA64, were cloned in temperate B. subtilis phage p11, and the amyR2 and amyE+ genes were then recloned in plasmid pUB110, which was designated pTUB4. The order of the restriction sites, ClaI-EcoRI-PstI-SalI-SmaI, found in the DNA fragment carrying amyR2 and amyE+ from the phage genome was also found in the 2.3-kilobase insert of pTUB4. Approximately 2,600 base pairs of the DNA nucleotide sequence of the amyR2 and amyE+ gene region in pTUB4 were determined. Starting from an ATG initiator codon, an open reading frame was composed of a total 1,776 base pairs (592 amino acids). Among the 1,776 base pairs, 1,674 (558 amino acids) were found in the cloned DNA fragment, and 102 base pairs (34 amino acids) were in the vector pUB110 DNA. The COOH terminal region of the alpha-amylase of pTUB4 was encoded in pUB110. The electrophoretic mobility in a 7.5% polyacrylamide gel of the alpha-amylase was slightly faster than that of the parental alpha-amylases. The NH2 termination portion of the gene encoded a 41-amino acid-long signal sequence (Ohmura et al., Biochem. Biophys. Res. Commun. 112:687-683, 1983). The DNA sequence of the mature extracellular alpha-amylase, a potential RNA polymerase recognition site and Pribnow box (TTGATAGAGTGATTGTGATAATTTAAAAT), and an AT-rich inverted repeat structure which has free energy of -8.2 kcal/mol (-34.3 kJ/mol) were identified. The AT-rich inverted repeat structure seemed to correspond to the hyperproducing character. The nucleotide sequence around the region was quite different from the promoter region of the B. subtilis 168 alpha-amylase gene which was cloned in the Escherichia coli vector systems.     

Cloning and expression of amyE gene from Bacillus subtilis in Zymomonas mobilis and direct production of ethanol from soluble starch

Two proven secretion signal zmo130 and zmo331 native to Zymomonas mobilis were fused to the N terminal of ??-amylase from Bacillus subtilis and transformed into 5 different strains of Z. mobilis separately. It was found that the signal zmo130 could direct the extracellular secretion of the expressed ??-amylase with high activity, but zmo331 could not. Fermentation experiments demonstrated that the recombinant Z. mobilis CICC 10225(p130A) exhibited the highest level of ethanol production, which is nearly 50% of the theoretical yield of ethanol from soluble starch, but another recombinant Z. mobilis ATCC 31821(p130A) took the shortest fermentation time of approximately 3 days, with the second high level of ethanol yield. The recombined strains in our study could be an important target for the following genetic engineering of next amylase in order to hydrolyze starch completely.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 A resolution

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-Amylase II (TVAII) has been determined by multiple isomorphous replacement at 2.6 A resolution. TVAII was crystallized in an orthorhombic system with the space group P212121 and the cell dimensions a=118.5 A, b=119.5 A, c=114.5 A. There are two molecules in an asymmetric unit, related by the non-crystallographic 2-fold symmetry. Diffraction data were collected at 113 K and the cell dimensions reduced to a=114.6 A, b=117.9 A, c=114.2 A, and the model was refined against 7.0-2.6 A resolution data giving an R-factor of 0.204 (Rfree=0.272). The final model consists of 1170 amino acid residues (two molecules) and 478 water molecules with good chemical geometry. TVAII has three domains, A, B, and C, like other alpha-amylases. Domain A with a (beta/alpha)8 barrel structure and domain C with a beta-sandwich structure are very similar to those found in other alpha-amylases. Additionally, TVAII has an extra domain N composed of 121 amino acid residues at the N-terminal site, which has a beta-barrel-like structure consisting of seven antiparallel beta-strands. Domain N is one of the driving forces in the formation of the dimer structure of TVAII, but its role in the enzyme activity is still not clear. TVAII does not have the Ca2+ binding site that connects domains A and B in other alpha-amylases, rather the NZ atom of Lys299 of TVAII serves as the connector between these domains. TVAII can hydrolyze cyclodextrins and pullulan as well as starch. Based on a structural comparison with the complex between a mutant cyclodextrin glucanotransferase and a beta-cyclodextrin derivative, Phe286 located at domain B is considered the residue most likely to recognize the hydrophobic cavity of cyclodextrins. The active-site cleft of TVAII is wider and shallower than that of other alpha-amylases, and seems to be suitable for the binding of pullulan which is expected not to adopt the helical structure of amylose.     

蜡样芽胞杆菌蛋白酶的克隆及在枯草芽胞杆菌系统中的高效表达

基于来源于Bacillus cereus WQ9-2的耐有机溶剂蛋白酶WQ的液相色谱-双质谱(LC-MS-MS)分析结果,设计引物,克隆耐有机溶剂蛋白酶WQ的基因,测序分析表明该蛋白酶的开放阅读框(ORF)大小为1 701 bp,编码566个氨基酸,其中含有信号肽(28个氨基酸)、前肽(220个氨基酸)及成熟肽序列(含954 bp编码318个氨基酸),相对分子质量约为3.7×104。将不带自身信号肽的蛋白酶基因apr WQ插入穿梭质粒p MA5中,构建了表达载体p MA5/apr WQ。该表达载体导入枯草芽胞杆菌WB600中获得阳性重组子WB600-p MA5/apr WQ,通过优化培养基成分以及培养条件,重组子发酵产蛋白酶体积酶活达到17 400 U/m L,约为高产野生菌产酶量的5倍。重组蛋白酶在多种有机溶剂(体积分数为50%)中表现出了良好的耐受性,验证了重组菌表达的蛋白酶与来源于B.cereus WQ9-2的耐有机溶剂蛋白酶性质一致,该耐有机溶剂蛋白酶的高效表达为进一步发挥其高效生物催化作用等实际应用奠定了基础。     

Isolation of 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine from a riboflavin-adenine-deficient mutant of Bacillus subtilis.

Studies were carried out to determine possible intermediates involved in the biosynthetic pathway of riboflavin, using resting cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis AJ1988. The cells excreted 6,7-dimethyl-8-ribityllumazine, the end product in the biosynthetic pathway, into the incubation medium in large amounts. The addition of glyoxal caused a large accumulation of a green fluorescent compound; an inverse relation was observed between the formation of the lumazine and the concentration of glyoxal. Furthermore, added [2-14C]guanine effectively incorporated into the lumazine and the fluorescent compound in the same specific activity during incubation. The fluorescent compound was isolated, purified, and identified by paper chromatographic, fluorometric, and spectrophotometric analyses. It was proved to be 8-(1'-D-ribityl)lumazine, which appeared to have been formed by a reaction between glyoxal and a possible intermediate in the cells. Accordingly, 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine was concluded to be an immediate precursor of 6,7-dimethyl-8-ribityllumazine.     

Amplification and deletion of the amyE+-tmrB+ gene region in a Bacillus subtilis recombinant-phage genome by the tmrA7 mutation.

A 22.4-kilobase DNA fragment containing the tmrA7-amyR2-amyE+-tmrB+-aroI+ region of the Bacillus subtilis N7 chromosomal DNA was cloned into a recombinant B. subtilis bacteriophage, p11-AA248. The amyE+-tmrB+ gene region, approximately 12.6 kilobases, in the phage genome was amplified in a tunicamycin-resistant (Tmr) Amy+ AroI+ transductant of B. subtilis by p11-AA248. On the other hand, the amyE+-tmrB+ region in the genomes of 80 to 90% of the phage particles was deleted when the phages were induced from the Tmr Amy+ AroI+ transductants by treatment with 1.0 micrograms of mitomycin C per ml. From analyses of the physical maps and DNA nucleotide sequences in the junction region of the deleted phage genome and the parental DNA fragments, it is suggested that the deletion occurred within a direct repeat sequence composed of 18 base pairs. The endpoints of the amplified gene region seemed to be closely related to both terminal regions of the deleted DNA.     

A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).