Molecular cloning and heterologous expression of pea seedling copper amine oxidase

The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum). The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P. sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface. A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast alpha-factor secretion signal. Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme. Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions. These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast.     

Biosynthesis of avermectins and lipids in Streptomyces avermitilis

Abstract The gene coding for a β- d -xylosidase (E.C. 3.2.1.37) of the thermophile Caldocellum saccharolyticum was isolated previously as part of a gene cluster which has been cloned in Escherichia coli . The enzyme characteristics were determined in E. coli using toluenized cell extracts. The pH optimum was 6.5 and temperature optimum 70°C. The enzyme was stable at 60°C and the half life at 80°C was 45 minutes. The temperature optimum and the temperature stability exceed those reported for other bacterial or fungal β- d -xylosidases. The enzyme showed no other detectable xylanolytic or cellulolytic enzyme activity.     

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7.

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.     

Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli.

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.     

Overexpression, purification and characterization of the Trichoderma atroviride endochitinase, Ech42, in Pichia pastoris

The endochitinase gene ech42 from Trichoderma atroviride was cloned and expressed in Pichia pastoris using a constitutive expression system. Over 98% of the recombinant protein was secreted into the culture medium as glycoprotein. A high endochitinase concentration, 186 mg/L with a specific enzyme activity of 14,128 Umg(-1) was produced. The optimal enzyme kinetic parameters for the recombinant protein were identical to those reported for the enzyme isolated from T. atroviride. The recombinant endochitinase possesses suitable features for biotechnological applications, such as activity at acidic pH and thermostability.     

Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.     

水稻ADP—葡萄糖焦磷酸化酶的cDNA基因克隆和结构分析

用ＰＣＲ技术从我国水稻品种“中花１０ 号”（Ｏｒｙｚａ ｓａｔｉｖａ ｖａｒ． ｊａｐｏｎｉｃａ ｃｖ． Ｚｈｏｎｇｈｕａ １０）的未成熟种子中克隆到ＡＤＰ－葡萄糖焦磷酸化酶基因,进行了全序列分析,并与国外已报道的同类基因进行了核苷酸及推导氨基酸序列的同源性比较。结果表明,作者克隆的ＡＤＰ－葡萄糖焦磷酸化酶基因全长１４６１ ｂｐ,编码１个由４８３ 个氨基酸组成的多肽,与国外基因的核苷酸和推导氨基酸同源率分别为９９．６％ 和９９．７％ 。此外,还对该基因进行了结构及进化分析。     

cDNA Cloning and Structural Analysis of Rice ADP-Glucose Pyrophosphorylase Gene

ADP-glucose pyrophosphorylase is a key enzyme in the pathway of starch synthesis. The gene encoding this.important enzyme was cloned by PCR amplification from immature seeds of a Chinese rice cultivar-Oryza sativa var. japonica cv. Zhonghua 10. It was then sequenced and compared with the same gene reported from other rice cultivar. The gene obtained in this study is composed of 1461 bp and encodes 483 amino acid residues. Its nucleotide sequence and deduced amino acid sequence shared 99.6% and 99.7% homology with those of published data respectively. The analyses on the structure and evolution of this gene have been conducted.     

Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene.

Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined. The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed. The enzymatic properties of the S. bovis malic enzyme were almost identical to those of other malic enzymes previously reported. However, we found that the S. bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate. The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity. A sequence analysis of the cloned fragment revealed the presence of two open reading frames that were 1,299 and 1,170 nucleotides long. The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis.     

Molecular cloning and characterization of an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside from Bacillus stearothermophilus SA0301

Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl alpha-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397-400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k(0)/K(m) values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s(-1).mM(-1) respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k(0)/K(m) value for isomaltose was 0.81 s(-1).mM(-1). The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.     

Molecular characterisation of the gene encoding an esterase from Bacillus licheniformis sharing significant similarities with lipases

An esterase gene from the moderate thermophilic strain Bacillus licheniformis LCB40 was cloned and expressed in Escherichia coli. Comparison of the amino acid sequence of the esterase with those of known lipases and esterases showed the presence of the well-conserved Gly-X-Ser-X-Gly pentapeptide, with an alanine replacing the first glycine. This substitution has never been reported for an esterase but it is present in the lipases from Bacillus subtilis, Bacillus pumilus and Galactomyces candidum. The amino acid sequence showed similarities with lipases and with mammalian lecithin-cholesterol acyltranferases and no similarities with esterases. The enzyme activity of a crude extract from a recombinant Escherichia coli strain showed hydrolysis of p-nitrophenyl caprylate (pNPC8) as for esterases, but not of p-nitrophenyl palmitate (pNPC16) or olive oil such as for lipases. Thus, the enzyme displays the original property of associating the activity of an esterase with a primary sequence showing high similarity with lipases.     

Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone.

6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum.     

Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K(m)) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

Arthrobacter nitroguajacolicus腈水解酶基因的克隆和表达

腈水解酶是一类能将腈类化合物催化生成酸的氰基水解酶。目前已有多个菌种的腈水解酶基因序列被报道,如敏捷食酸菌Acidovoraxfacilis,粪产碱菌Alcaligenesfaecalis,睾丸丛毛单胞菌Comamonastestoteroni,肺炎克雷伯菌Klebsiellapneumoniae,假单胞菌Pseudomonas属菌株,红球菌Rhodococcus属菌株,但节杆菌属菌株Arthrobacternitroguajacolicus的腈水解酶基因序列尚未见报道。经由野生型酶的分离纯化,基因文库筛选及侧翼序列扩增等步骤,克隆得到该菌株的腈水解酶基因,从而为进一步研究该酶的特性及构建用于工业生产的重组菌打下基础。     

Structure of bovine adrenal dopamine beta-monooxygenase, as deduced from cDNA and protein sequencing: evidence that the membrane-bound form of the enzyme is anchored by an uncleaved signal peptide

A full-length cDNA for dopamine beta-monooxygenase (D beta M) from bovine adrenal glands has been cloned and sequenced. The soluble and membrane-derived forms of D beta M have also been sequenced from their N-termini. While the observed sequences for the soluble protein correspond to those previously reported [Joh, T.H., & Hwang, O. (1986) Ann. N.Y. Acad. Sci. 493, 343-350], the heavy subunit of membrane-derived enzyme is found to contain a unique N-terminus. Alignment of this N-terminus with that deduced from cDNA cloning indicates identity at 22 (and possibly 26) out of 27 residues. This comparison leads us to conclude that the membranous form of bovine D beta M retains an uncleaved N-terminal signal peptide as the source of membrane anchoring.