Isomaltulose synthase from Klebsiella sp. strain LX3: gene cloning and characterization and engineering of thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an alpha-amylase domain and (beta/alpha)(8)-barrel structures, suggesting that it belongs to the alpha-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in alpha-amylases and glucosyltransferases (Asp(241), Glu(295), Asp(369), His(145), and His(368)) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 +/- 1.7 mM for sucrose, and maximum activity (approximately 328.0 +/- 2.5 U/mg) at pH 6.0 and 35 degrees C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50 degrees C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu(498) and Arg(310) with proline resulted in an 11-fold increase in the half-life of PalI at 50 degrees C.     

Isomaltulose synthase (PalI) of Klebsiella sp. LX3. Crystal structure and implication of mechanism

Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) and trehalulose (alpha-D-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-A resolution with an R-factor of 19.4% and Rfree of 24.2%, consists of three domains: an N-terminal catalytic (beta/alpha)8 domain, a subdomain between N beta 3 and N alpha 3, and a C-terminal domain having seven beta-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology.     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ESO109中分离到一株具有高内切葡聚糖酶活力的细菌DY3,16SrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp．)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99％。PCR扩增DY3的内切葡聚糖酶基因celX全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Rseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95％的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40℃,酶的最适pH在6～7之间。     

Cloning of a Novel Pyrethroid-Hydrolyzing Carboxylesterase Gene from Sphingobium sp. Strain JZ-1 and Characterization of the Gene Product

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many α/β-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.Pyrethroid pesticides are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic organophosphorus pesticides, and their usage is continuing to grow (10). Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate insecticides, exposure to very high levels of pyrethroid pesticides might cause endocrine disruption, lymph node and spleen damage, and carcinogenesis (6, 12). In addition, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, often at concentrations of less than 0.5 μg/kg (19, 22). Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment.In general, pyrethroid pesticides are degraded by both abiotic and biotic pathways, including photooxidation, chemical oxidation, and biodegradation. Microorganisms play the most important role in degradation of pyrethroids in soils and sediments. Many pyrethroid-degrading microorganisms have been isolated from soils (13, 16, 24, 27).The major routes of pyrethroid metabolism in pyrethroid-resistant insects and pyrethroid-degrading microorganisms include oxidation by cytochrome P450s and ester hydrolysis by carboxylesterases (9). Carboxylesterases are a family of enzymes that are important in the hydrolysis of a large number of endogenous and xenobiotic ester-containing compounds, such as carbamates, organophosphorus pesticides, and pyrethroids. Carboxylesterases from Bacillus cereus SM3 (17), Aspergillus niger ZD11 (13), Nephotettix cincticeps (2), and mouse liver microsomes (23) hydrolyzing the carboxyl ester linkage of the pyrethroids were purified to homogeneity and characterized. Genes encoding the pyrethroid-hydrolyzing carboxylesterases from mouse liver microsomes and Klebsiella sp. strain ZD112 were cloned and functionally expressed (23, 27).Pyrethroids differ from many other pesticides in that they contain one to three chiral centers; the chirality may arise from the acid moiety, the alcohol moiety, or both (Fig. ​(Fig.1).1). A pyrethroid compound therefore consists of two to eight isomers. Isomers of a chiral compound often differ from each other in biological properties. Isomer selectivity has been widely observed in insecticidal activity for the isomers of a pyrethroid compound. Recently, studies have shown that biodegradation of pyrethroids also exhibits significant isomer selectivity (15, 23).Open in a separate windowFIG. 1.Molecular structures of pyrethroids tested. Chiral centers are indicated by black dots.In this study, we described the isolation and identification of the pyrethroid-degrading Sphingobium sp. strain JZ-1, the cloning and expression of the gene pytH encoding a novel pyrethroid-hydrolyzing carboxylesterase, and the characterization of the purified enzyme.     

海藻糖合酶产生菌筛选、鉴定及目的基因克隆

目的:为筛选出一株产海藻糖合酶的菌株,并以此菌的全DNA为模板,克隆出产海藻糖合酶的目的基因片段。方法:实验过程中采用了常规筛选菌种、快速提取细菌全基因、显微镜观察菌种、热启动PCR技术、电泳纯化回收基因片段、EcoRⅠ和HindⅢ双酶切鉴定目的基因片段等方法。结果:在电镜下可观察到有芽孢、杆菌;菌株16S rRNA基因扩增产物共计1490个碱基;PCR方法扩增出阳性克隆大约1700bp的基因片段。结论:通过生理、形态、结构特征分析及16S rRNA基因全序列比较得出结论:筛选到一株短小芽孢杆菌;PCR扩增出阳性克隆片段,全长1722bp,为实验所要的编码海藻糖合酶的基因片段。     

Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene from Vibrio sp. Strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40°C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.     

小苍兰ACC合成酶FhACS1基因的克隆与序列分析

以小苍兰品种“上农金皇后”为研究对象,利用PCR技术和染色体步移技术首次克隆到了ACC合成酶FhACS1基因的全长序列和编码序列。结果表明：FhACS1基因全长为2 576 bp,包含长为433 bp的5′端非编码区序列（5′-UTR）以及长为373 bp的3′端非编码区序列（3′-UTR）;开放读码框（ORF）长度为1 371 bp,推定编码457个氨基酸。分析表明,该基因包含3个内含子和4个外显子。序列分析结果显示,FhACS1推导蛋白具备ACS蛋白的7个保守结构域;在氨基酸水平上,FhACS1与小果芭蕉的同源性最高（85%）;系统进化分析表明,FhACS1与孟宗竹BaACS（BAC56949.1）、水稻OsACS1（AAA33888.1）、小果芭蕉MaACS1（AAQ13435）的亲缘关系最近。在其已知启动子区域,发现有多个对脱落酸、赤霉素、细胞分裂素等激素响应的顺式元件,以及对干旱和低温等逆境胁迫响应的顺式元件。     

野葛异黄酮合酶基因的分离与功能验证

异黄酮是野葛（Pueraria lobata）中的主要活性成分,而异黄酮合酶（IFS）是催化异黄酮生物合成的第一步关键酶,尽管野葛的IFS基因已被分离,但其功能还未得到任何验证。本研究以中国安徽省郎溪县的野葛为材料,利用RT-PCR技术成功克隆到野葛IFS基因,命名为PlIFS,PlIFS开放阅读框大小为1566 bp,编码521个氨基酸,将该基因克隆到GAL1启动子控制下的酵母表达载体pESC-TRP上,得到重组质粒pESC-TRP-PlIFS,通过LiAc/ssDNA/PEG方法将其转化进酿酒酵母（Saccharomyces cerevisiae）WAT11中进行异源表达,并在酵母体内对其活性进行验证,结果显示PlIFS能催化甘草素生成大豆苷元,表现出异黄酮合酶活性特征。荧光定量PCR分析显示,PlIFS基因主要在野葛的根中表达,这与活性物质异黄酮主要在野葛根中的积累模式一致。     

Thermostable Chitosanase from Bacillus sp. Strain CK4: Cloning and Expression of the Gene and Characterization of the Enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80°C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Substrate induction of isomaltulose synthase in a newly isolated Klebsiella sp. LX3

AIMS: Production of isomaltulose by newly isolated Klebsiella sp. LX3. METHODS AND RESULTS: The bacterial isolate LX3, which transforms sucrose to isomaltulose and trehalulose, has been isolated from a soil sample in Singapore. Morphological and biochemical analysis, as well as 16s rRNA sequence demonstrated that the isolate could represent a new member of genus Klebsiella. The strain has several interesting features. The immobilized cells of Klebsiella sp. LX3 convert more than 99% of sucrose to products that consist of more than 87% of isomaltulose, 11.6% of trehalulose, and <1% of glucose. CONCLUSIONS: The production of isomaltulose synthase in isolate LX3 is inducible by its substrate sucrose and the sugars containing a fructofuranosyl group. SIGNIFICANCE AND IMPACT OF STUDY: It would be useful for future biotechnological applications to understand the structural features or motifs of the isomaltulose synthases that determine the sucrose conversion efficiency and the ratio of the conversion products.     

Sucrose-Phosphate Synthase from Synechocystis sp. Strain PCC 6803: Identification of the spsA Gene and Characterization of the Enzyme Expressed in Escherichia coli

The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.     

Characterization of Porphobilinogen Synthase from an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

Porphobilinogen synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) was purified 7,405-fold from an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114. The molecular weightof the enzyme was determined to be 260,000 by Sephadex G-200gel filtration. The enzyme had a single pH optimum at 8.0 andshowed no requirement for metal ion and thiol compound for itsmaximum activity. The K_m value for 5-aminolevulinic acid was0.29 mM. 4,5-Dioxovaleric acid and levulinic acid were foundto be competitive inhibitors of the enzyme, with K_i values of0.65 and 0.80 mM, respectively. The enzyme was extremely labilein acidic pH and almost completely lost its activity within1 h at pH 6.0 and 30?C. This Erythrobacter enzyme seems to besimilar to the enzyme from the anaerobic photosynthetic bacteriumRhodobacter capsulatus in its molecular and catalytic properties. (Received February 17, 1988; Accepted May 9, 1988)     

南极耐冷 Pseudomonas sp. 7323低温脂肪酶的性质及基因克隆

 南极微生物是筛选低温酶的良好来源,但尚未得到充分的研究与开发.低温脂肪酶具有广阔的应用前景,其基因结构特征也具有重要的研究意义. 本文对南极微生物开展了低温脂肪酶产生菌的筛选、基因克隆及特征分析.采用功能筛选的方法,从南极普里兹湾深海沉积物中获得一株产低温脂肪酶的菌株7323,其最适温度和最高生长温度分别为20℃和30℃,属于耐冷菌.16S rDNA序列分析表明,该菌属于假单胞菌属（Pseudomonas）.通过设计引物扩增出的脂肪酶基因全长为1854　bp,该基因编码一个由617氨基酸、分子量预计为64466的蛋白质.氨基酸序列分析表明,该酶与Pseudomonas sp. UB48 的脂肪酶有89%的相似性,在催化区和C末端信号肽中存在高度保守的序列.纯化后的酶学性质研究表明,该脂肪酶的最适温度为35℃,最适pH值为9.0,为碱性低温酶.     

Cloning and Characterization of a Gene Cluster for Hatomarubigin Biosynthesis in Streptomyces sp. Strain 2238-SVT4

Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.The angucycline antibiotics are a large group of naturally occurring aromatic polyketides of microbial origin (11, 15). They exhibit a wide range of biological activities, which include antibacterial, antiviral, antitumor, enzyme inhibitory, and platelet aggregation inhibitory effects. Although all the members contain a benz[a]anthraquinone skeleton of decaketide origin, their structural diversity is very broad and they have a wide variety of oxidation states. Hatomarubigins A, B, C, and D (Fig. ​(Fig.1)1) belong to the angucycline family and reverse colchicine resistance in multidrug-resistant tumor cells (8). Among them, hatomarubigin D is a unique hatomarubigin C dimer with a methylene linkage. Such a dimer has not been reported previously, and little is known about the mechanism of the methylene bridge formation between two aromatic rings. In this study, a gene cluster for hatomarubigin biosynthesis was identified in Streptomyces sp. strain 2238-SVT4, and a part of the gene cluster was expressed in Streptomyces lividans to produce the hatomarubigins.Open in a separate windowFIG. 1.Structures of angucycline antibiotics.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Gene Cloning,Nucleotide Sequencing,and Purification and Characterization of the Low-Specificity l-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictly l specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, including l-β-3,4-dihydroxyphenylserine, l-β-3,4-methylenedioxyphenylserine, and l-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity l-TA from Saccharomyces cerevisiae, l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the l-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.β-Hydroxy-α-amino acids constitute an important class of compounds. They are natural products in their own right and are components of a range of antibiotics, for example, cyclosporin A, lysobactin, and vancomycin (30) and bouvardin and deoxybouvardin (6). 4-Hydroxy-l-threonine is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5′-phosphate (PLP)-dependent enzymes (32). 3,4,5-Trihydroxyl-l-aminopentanoic acid is a key component of polyoxins (32). l-threo-3,4-Dihydroxyphenylserine is a new drug for Parkinson’s disease therapy (13). However, the industrial production of β-hydroxy-α-amino acids has been limited to chemical synthesis processes, which need multiple steps to isolate the four isomers (l-threo form, d-threo form, l-erythro form, and d-erythro form). Threonine aldolase (EC 4.1.2.5), which stereospecifically catalyzes the retro-aldol cleavage of threonine, is a potentially useful catalyst for the synthesis of substituted amino acids from aldehyde and glycine (27, 31, 32).Two different types of threonine aldolases are known so far. l-allo-Threonine aldolase (l-allo-TA), isolated and purified from Aeromonas jandaei DK-39 (8), stereospecifically catalyzes the reversible interconversion of l-allo-threonine and glycine. Low-specificity l-threonine aldolase (l-TA) catalyzes the cleavage of both l-threonine and l-allo-threonine to glycine and acetaldehyde, as well as the reverse reaction, aldol condensation. The enzymes have been purified and characterized from Candida humicola (9, 34) and Saccharomyces cerevisiae (12). Low-specificity l-TA activity has also been shown to exist in mammals (7, 23, 26) and a variety of other microbial species (2, 4, 17, 35). The enzyme is physiologically important for the synthesis of cellular glycine in yeast (12, 15, 16). Threonine aldolases with distinct stereospecificities are ideal targets for enzymology studies on structural and functional relationships. However, information on the primary structures of threonine aldolases was limited to our recent studies (11, 12). The construction of an overproduction system for threonine aldolase will be indispensable for the industrial biosyntheses of β-hydroxy-α-amino acids.The present work focuses on the cloning, sequencing, and overexpression in Escherichia coli cells of the low-specificity l-TA gene from Pseudomonas sp. strain NCIMB 10558, the purification and characterization of the recombinant enzyme, and the identification of the active-site lysine residue of the enzyme by site-directed mutagenesis. Evidence is presented that Lys207 of low-specificity l-TA probably functions as a catalytic residue, forming an internal Schiff base with the PLP of the enzyme to catalyze the reversible aldol reaction. This is the first report showing a purified enzyme with l-β-3,4-dihydroxyphenylserine aldolase and l-β-3,4-methylenedioxyphenylserine aldolase activities, providing a new route for the industrial production of these important unnatural amino acids.     

Cloning and Characterization of Extracellular Metal Protease Gene of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.     

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His₆-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K_m and k_cat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min⁻¹, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.     

A Maltooligosaccharide-Forming Amylase Gene from Brachybacterium sp. Strain LB25: Cloning and Expression in Escherichia coli

Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.