Purification and characterization of the lipase from Pseudomonas fluorescens HU380

A lipase, which markedly splits polyunsaturated fatty acid ester (PUFA) bonds, from newly isolated Pseudomonas fluorescens HU380 was purified. The purification procedure included Phenyl-Toyopearl fractionation, DEAE-Sepharose chromatography, and Superdex-200HR chromatography. The enzyme was purified 24.3-fold with a yield of 14% and a specific activity of 9854 U/mg. Its molecular weight was estimated on SDS-PAGE to be 64,000. The optimum pH and temperature were 8.5 and 45 degrees C, respectively. The lipase was stable over the pH range of 6.0-7.0 at 30 degrees C for 24 h, and up to 40 degrees C at pH 7.0 for 60 min, when 0.1% Triton X-100 was present. The lipase preferably acted on short to middle-chain fatty acid simple methyl-esters and triglycerides, and cleaved mainly 1,3-ester bonds and to a lesser extent the 2-position ester bond of triolein. The lipase was inhibited by Co2+, Ni2+, Fe3+, Fe2+, and EDTA, and activated by Ca2+. Its N-terminal amino acid sequence was determined to be GVYDYKNFGTADSKALFSDAMAITLY, which exhibited considerable similarity with those of the lipases from other P. fluorescens strains, but no significant homology with other lipases. This lipase was able to decompose fats and oils that contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) without significantly affecting the contents of these fatty acids. The results suggest that the lipase may be useful when applied to the processing of industrial fats and oils containing EPA and DHA, such as fish oil splitting.     

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.     

Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles

The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat. Lipases were isolated from a shaken skim milk culture of P. fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150. The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity. Disc gel electrophoresis of partially purified enzyme revealed two protein bands. These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil. Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography. Data from gas chromatographic analysis indicated that P. fluorescens 27 produces at least two different lipases.     

Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them

P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14-34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.     

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 (Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80 degrees C. However, the protein showed little GTPase activity at 40 degrees C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [alpha-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42 degrees C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90 degrees C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.     

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.     

Cloning and sequencing of the histidine decarboxylase gene from Photobacterium phosphoreum and its functional expression in Escherichia coli

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5'-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.     

荧光假单胞菌胞外蛋白酶的纯化及特性研究

采用硫酸铵沉淀、DEAE-Sepharose FF离子交换层析、Sephacryl S-100 HR凝胶过滤层析方法对从原料乳中分离出一株荧光假单胞菌胞外耐热蛋白酶进行纯化及特性研究。将纯化后的单一蛋白酶进行SDS-PAGE分子质量测定,并考察最适温度、最适pH值、热稳定性、金属离子对其酶活性的影响及对该蛋白酶的氨基酸种类分析。纯化后蛋白酶的分子质量为47kD,经纯化后蛋白酶比活力提高了近61.38倍;最适温度和pH值分别为30℃和7.0;二硫苏糖醇对蛋白酶活性具有一定的抑制作用,Fe2+可以促进蛋白酶活性的提高;该酶具有较强耐热性,原酶液经130℃热处理3min后,残留酶活仍超过原酶液的47.67%;该蛋白酶氨基酸组成中甘氨酸(Gly)、丙氨酸(Ala)和谷氨酸(Glu)的含量占明显优势,其中Gly含量最高,物质的量分数高达42%。     

Cloning and expression of chitin deacetylase gene from a Deuteromycete, Colletotrichum lindemuthianum

The chitin deacetylase gene was cloned from cDNA of Colletotrichum lindemuthianum ATCC 56676, and the open reading frame consisted of a possible prepro-sequence of 27 amino acids at the N-terminus and a mature chitin deacetylase. The deduced amino acid sequence of the mature enzyme revealed 26% identity and 46% similarity with a chitin deacetylase from Mucor rouxii. The molecular mass of the protein estimated from the amino acid sequence data was 24.3 kDa, which was in good agreement with the MALDI-TOF MS analysis data of the purified protein (24.17-24.36 kDa). The gene product was overexpressed in Escherichia coli cells as a fusion protein with six histidine residues at its C-terminus. The fusion protein formed inclusion bodies, but chitin deacetylase activity was restored from the inclusion bodies by a simple renaturation step with 8 M urea treatment. The recombinant enzyme was purified by affinity chromatography and gel filtration steps, and had a final specific activity of 4.22 units mg(-1) of protein. Trypsin digestion of the recombinant enzyme resulted in 2.1-fold increase in activity, suggesting that the removal of the prepro-domain from the recombinant enzyme resulted in an increase in its activity.     

Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae

The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.     

Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme

A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.     

扬州稀甜酱中乳酸菌的分离鉴定及抑菌活性初探

以扬州稀甜酱为研究对象,采用经典乳酸菌筛选法从样品中分离、纯化乳酸菌,采用形态学观察、生理生化试验和16S rRNA序列分析对乳酸菌进行鉴定,并采用牛津杯法对发酵滤液的抑菌活性进行研究。结果表明,从扬州稀甜酱中共分离、纯化出33株乳酸菌,其中15株菌株被鉴定为屎肠球菌（Enterococcus faecium）,12株菌株被鉴定为乳酸片球菌（Pediococcus acidilactici）,4株菌株被鉴定为类肠膜魏斯氏菌（Weissella paramesenteroides）,2株菌株被鉴定为酸鱼乳杆菌（Lactobacillus acidipiscis）,屎肠球菌和乳酸片球菌为扬州稀甜酱中的优势乳酸菌。乳酸片球菌TJ17和TJ31发酵滤液对金黄色葡萄球菌（Staphylococcus aureus）、单核细胞增生李斯特氏菌（Listeria monocytogenes）和大肠埃希氏菌（Escherichia coli）均有明显的抑制作用,且乳酸片球菌TJ31抑菌效果较好。     

草莓轻型黄边病毒外壳蛋白抗原表位预测、克隆、表达及其免疫原性分析

应用IEDB、DNAStar、DNAMAN和Snap Gene等生物信息学工具对草莓轻型黄边病毒外壳蛋白的氨基酸残基序列进行分析。选择一段抗原性较强的肽段(位于外壳蛋白27~38氨基酸残基处),依据大肠杆菌(Escherichia coli)的密码子偏好性化学合成了该肽段的DNA编码序列(AE)。AE片段克隆至E.coli表达载体pET32a(+)的Eco RⅠ和XhoⅠ位点,获得重组质粒pET32a(+)-AE。含AE片段的开放阅读框长561 bp,编码了一个187氨基酸残基的重组融合蛋白,理论分子质量为20.29kD。重组质粒pET32a(+)-AE分别在E.coli BL21(DE3)和E.coli Rosetta中进行了诱导表达和条件优化。重组融合蛋白在E.coli Rosetta中的最佳表达条件为1.5 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)、35℃诱导表达2 h,产率为13.22 mg/L;在E.coli BL21(DE3)中的最佳表达条件是为0.5 mmol/L IPTG、30℃诱导表达2 h,产率为9.55 mg/L。重组融合蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离、质谱鉴定表明,其含有所预测的草莓轻型黄边病毒外壳蛋白抗原表位肽段AE。AE重组融合蛋白经Ni~(2+)亲和色谱纯化、EK酶切、分子筛除去融合蛋白标签后,免疫产蛋鸡。从免疫鸡所产鸡蛋中提取鸡卵黄免疫球蛋白(immunoglobulin of yolk,Ig Y),Dot-Blot检测抗体结合活性。结果表明,所提取的IgY可特异性识别AE肽段和SMYEV外壳蛋白。这一结果表明所预测的AE肽段具有较好的免疫原性。     

Acidolysis and glyceride synthesis reactions using fatty acids with two Pseudomonas lipases having different substrate specificities

Enzymatic acidolysis and glyceride synthesis using polyunsaturated fatty acids (PUFAs) with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase) were studied. The acidolysis of triolein with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 degrees C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 degrees C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 degrees C, respectively. The EPA incorporation rate was even higher at a low temperature (10 degrees C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, arachidonic acid (AA), mead acid (MA), and dihomo-gamma-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e., MA, AA, EPA, and DHA, were low for both reactions. The unique substrate specificities of the lipases from the Pseudomonas strains will enable us to use these lipases for the modification of fats and oils containing PUFAs such as fish oil.     

Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.     

Cloning and expression of the N-acetylmuramidase gene from Streptomyces rutgersensis H-46

The N-acetylmuramidase SR1 gene from Streptomyces rutgersensis H-46 was cloned in Escherichia coli JM109 and expressed in E. coli BL21(DE3)pLysS. An open reading frame included the leader peptide region encoding a polypeptide of 65 amino acid residues and the mature SR1 enzyme region encoding a polypeptide of 209 amino acid residues. The overall G + C content of the mature enzyme gene was 67.6%, with 98.1% of G or C in the third position of the codons. The calculated molecular weight of the mature enzyme was 23,057 Da. The amino acid sequence of the mature enzyme showed a significant level of identity with bacteriolytic enzymes from Streptomyces globisporus (50.9% identity), Chalaropsis species (40.2% identity) and Saccharopolyspora erythraea (31.0% identity). The mature enzyme gene cloned into plasmid pET26b carrying a signal peptide, peIB, was expressed in E. coli BL21(DE3)pLysS. The signal peptide region was cleaved during the production of the enzyme. Specific activity of the enzyme purified from the transformant was almost identical to that of the native enzyme. Furthermore, the SR1 enzyme gene cloned with the leader peptide gene into plasmid pET28a was also expressed in E. coli. In this case, a proform-like protein was partially processed; 35 amino acid residues were cleaved but 30 amino acid residues remained. This proform like protein has approximately one-nineteenth the activity of the native enzyme. These results indicated that the native SR1 enzyme was produced in the following manner in the cells of S. rutgersensis H-46. The SR1 enzyme gene was translated to a pre-proform protein followed by the deletion of a signal peptide. Finally, the proform-like protein was processed by deletion of the remaining leader peptide.     

磷脂酶A1(Lecitase Ultra)的纯化及氨基酸序列分析

本论文研究了超滤法和RP-HPLC对磷脂酶A1(Lecitase Ultra)的纯化,利用MALDI SYNAPT Q-TOF MS和MS/MS对其氨基酸序列进行分析及推测。纯化去除了Lecitase Ultra中的山梨醇和山梨醇盐,纯度97.7%,酶蛋白和亚基的分子量为30838.0和16536.0。通过MALDI SYNAPT Q-TOF MS和MS/MS对酶蛋白进行分析,确定了Lecitase Ultra的氨基酸序列组成;进行数据库(http: //www.matrixscience.com)比对,发现其氨基酸序列信息与棉状嗜热丝孢菌(T. lanuginosus)脂肪酶和尖孢镰刀菌(F. oxysporum)脂肪酶高度吻合,序列中1-284是T. lanuginosus脂肪酶的氨基酸序列,序列中285-339是脂肪酶F. oxysporum脂肪酶的氨基酸序列,其中113、118和121可能是被基因突变成113 G-A、118 D-W和121 E-K。研究结果为进一步探讨Lecitase Ultra的三维空间结构及其活性中心催化机理提供了数据支撑。     

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.     

Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472

A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)K(m) value for xylitol was two and four times higher than those for galactitol and n-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65 degrees C. The optimal temperature and pH were 55 degrees C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 by that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X-Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications.