Cloning and heterologous expression of a beta-fructofuranosidase gene from Arthrobacter globiformis IFO 3062, and site-directed mutagenesis of the essential aspartic acid and glutamic acid of the active site

We have cloned the gene encoding a beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and subsequently, the gene was heterologously expressed in Escherichia coli. This beta-fructofuranosidase gene encodes a protein of 548 amino acid residues with a calculated molecular mass of 60,519 Da. We have examined the roles of three residues of A. globiformis IFO 3062 beta-fructofuranosidase by site-directed mutagenesis, and found that aspartic acid 130 and glutamic acid 392, which are two of the apparent catalytic residues, are essential for hydrolase activity. This study provides the first experimental evidence showing that these two amino acid residues of beta-fructofuranosidase play a critical role in hydrolyzing sucrose.     

Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product

Arthrobacter globiformis FERM BP-360 produces uricase (urate oxidase; EC 1.7.3.3) intracellularly. A genomic library of the bacterium, prepared in the plasmid vector pUC118, was screened with probes based on the amino acid sequence of the purified uricase. We found that a chimeric plasmid in the library, designated pUOD1, carries a 2.0-kb DNA insert from the Arthrobacter DNA that hybridizes with the probe. The DNA insert contains an ORF consisting of 302 amino acids with a calculated molecular mass of 33,858. The protein translated from the ORF displays the highest identity (67%) to uricase from a bacterium, Cellulomonas flavigena. X-ray fluorescence analysis showed that the Arthrobacter uricase contains copper ion. However, we found that the catalytic activity of uricase is inhibited by the excessive addition of copper ion. Although the production of A. globiformis uricase is induced by the addition of uric acid to the culture medium, Escherichia coli harboring pUOD1 produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.     

Preservation of acidified cucumbers with a combination of fumaric acid and cinnamaldehyde that target lactic acid bacteria and yeasts

The naturally occurring compound, fumaric acid, was evaluated as a potential preservative for the long-term storage of cucumbers. Fumaric acid inhibited growth of lactic acid bacteria (LAB) in an acidified cucumber juice medium model system resembling conditions that could allow preservation of cucumbers in the presence of sodium benzoate. Forty millimolars of fumaric acid were required to inhibit growth of an extremely aciduric Lactobacillus plantarum LA0445 strain at pH 3.8. Half of this concentration was required to achieve inhibition of L. plantarum LA0445 at pH 3.5. As expected growth of the spoilage yeasts Zygosaccharomyces globiformis and Z. bailii was not inhibited by fumaric acid at near saturation concentrations in the same cucumber juice medium. To usefully apply fumaric acid as a preservative in acidified foods it will be necessary to combine it with a food grade yeast inhibitor. The antimicrobial agent, cinnamaldehyde (3.8 mM) prevented growth of Z. globiformis as well as the yeasts that were present on fresh cucumbers. Acidified cucumbers were successfully preserved, as indicated by lack of yeasts or LAB growth and microbial lactic acid or ethanol production by a combination of fumaric acid and cinnamaldehyde during storage at 30 °C for 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate, potassium sorbate, or sodium metabisulfite for preservation of acidified vegetables without a thermal process. PRACTICAL APPLICATION: This study evaluates the potential application of alternative preservatives for the long-term storage of cucumbers in a reduced NaCl cover brine solution and without a thermal process.     

Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway

The KEX2 protease (product of the KEX2 gene) functions late in the secretory pathway of Saccharomyces cerevisiae by cleaving the polypeptide chains of prepro-killer toxin and prepro-alpha-factor at paired basic amino acid residues. The intracellular vesicles containing KEX2 protease sedimented in density gradients to a position distinct from those containing mannosyltransferase I (product of the MNN1 gene), a marker enzyme for the Golgi complex. The recovery of intact compartments containing these enzymes approached 80% after sedimentation. We propose that the KEX2 protease and mannosyltransferase I reside within distinct compartments.     

Molecular cloning of the gene encoding the di-D-Fructofuranose 1,2':2,3' dianhydride hydrolysis enzyme (DFA IIIase) from Arthrobacter sp. H65-7

The gene encoding an intracellular enzyme hydrolyzing di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) (DFA IIIase) was cloned from the genomic DNA of Arthrobacter sp. H65-7 for the first time. The single open reading frame (ORF) of the DFA IIIase gene consisted of 1368-bp encoding 455 amino acids. DFA IIIase showed a phylogenetically distinct position from other inulin-degrading enzymes and showed similarity only with inulin fructotransferases (depolymerizing) (inulase II, EC 2.4.1.93) from Arthrobacter globiformis C11-1, Arthrobacter sp. A-6, and Arthrobacter sp. H65-7 (48.7-50.3%), and inulin fructotransferase (DFA I-producing) (EC 2.4.1.200) from A. globiformis S14-3 (44.4%). An Escherichia coli transformant harboring a recombinant plasmid, pINB2, in which the DFA IIIase gene was fused with the beta-galactosidase of pUC19 and under the control of the lac promoter, expressed DFA IIIase and the cloned enzyme produced inulobiose from DFA III similarly to the DFA IIIase of the wild-type strain, Arthrobacter sp. H65-7.     

Influence of Microbial Growth on the Redox Potential of Fermented Cucumbers

ABSTRACT:  Commonly, pH measurements are used during the production of fermented cucumbers to indirectly monitor growth of lactic acid bacteria (LAB) and acid production. Redox potential (E_h) measurements, which are determined by the potential of an electron to reduce an acceptor, could serve as an alternative tool to monitor the progress of fermentation allowing the detection of the metabolic activity and/or growth of LAB and other microorganisms. Pasteurized and inoculated jars of cucumbers were observed to better understand how the E_h changes during the cucumber fermentation and how it could be used as a monitoring tool. Jars of diced, brined cucumbers were pasteurized and inoculated with microbes previously isolated from fermented cucumbers including Lactobacillus plantarum , Zygosaccharomyces globiformis , and Enterobacter aerogenes . Although an initial decrease in E_h was observed for all microorganisms, distinctive trends in E_h occurred when these organisms were inoculated. After a 2-wk fermentation period, the E_h (Ag/AgCl, 3 M KCl) in jars inoculated with L. plantarum , Z. globiformis , and E. aerogenes was at +453 ± 55, +104 ± 5, and –156 ± 73 mV, respectively. Cucumbers inoculated with a mixture of L. plantarum and Z. globiformis had a terminal E_h value of +202 ± 24 mV, which was between that found for the individual microorganisms. L. plantarum dominated the E_h trend when inoculated along with E. aerogenes with a final E_h of +411 ± 72 mV. The results showed that changes in E_h continued after pH measurements became stable. Thus E_h measurement can provide a tool to continuously monitor microbial growth during the course of cucumber fermentations.     

Comparative studies on protein fractions and amino acid composition from sorghum and pearl millet

A defatted flour sample of sorghum and pearl millet were separated into three fractions. These procedures involve extracting the defatted flour with aqueous sodium hydroxide (pH 11.9) followed by precipitation with diluted HCl acid (pH 4.8). The two protein fractions I (soluble at pH 4.8) and II (insoluble at pH 4.8) along with the remaining residues (fraction III) were lyophilized separately. The amino acid composition of the original flour and the three fractions were determined. Lysine seems to be the most deficient amino acid in the original flour and the remaining residues. Fraction I and II, in which the lysine accumulated, have essentially better amino acid profile and consequently nutritionally better quality than the protein of the original defatted flour. The recovered protein-rich fractions I and II should be useful as a protein ingredient in foods. The remaining residues can be extruded into convenience foods.     

球形节杆菌C224分批发酵2-酮基-D-葡萄糖酸的条件优化及其动力学模型的构建

通过优化发酵条件,提高球形节杆菌C224菌株利用大米淀粉水解糖分批发酵2-酮基-D-葡萄糖酸的生产强度,并在此基础上构建其发酵动力学模型。在50 L的发酵罐上考察了通气量与葡萄糖浓度对2-酮基-D-葡萄糖酸发酵的影响,并基于Logistic方程、Luedeking-Piret方程和物料平衡计算分别构建了菌体生长、产物形成和底物消耗的动力学模型。研究结果表明,通气量低于1.5 v.v-1.m-1时,发酵生产强度明显减小,糖酸转化率有所降低;发酵培养基中的葡萄糖浓度以120.0～180.0 g/L为宜,过高或过低对生产强度和糖酸转化率均有不利影响。在适宜的发酵条件下,球形节杆菌C224菌株分批发酵生产2-酮基-D-葡萄糖酸的生产强度达6.36～6.54 g/(L.h),糖酸转化率为0.96～0.97 g/g。所构建动力学模型的计算值与实验值拟合效果良好,各项模型的相关系数R2均大于0.95,说明其能够揭示球形节杆菌C224分批发酵2-酮基-D-葡萄糖酸代谢基本特征。     

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.     

Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions I and IV of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the lcc1 gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences.     

2-酮基-D-葡萄糖酸产生菌球状节杆菌K1022抗噬菌体菌株的选育

从 2 酮基 D 葡萄糖酸产生菌球状节杆菌K10 2 2的异常发酵液中分离到 3种噬菌体 ,分别将其命名为KS2 11、KS2 12和KS2 13。采用紫外诱变或自然选育的方法 ,获得了 6株具有抗噬菌体能力的 2 酮基 D 葡萄糖酸高产菌株。研究了抗株C2 2 4和K2 32的遗传稳定性 ,并在 5 0kL的发酵罐上进行了发酵生产试验。结果表明 ,经多次传代 ,菌株C2 2 4和K2 32对噬菌体的抗性及其发酵产酸能力没有改变 ,可以应用于工业生产中     

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix)

BACKGROUND: As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. RESULTS: Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE‐Sepharose and Superdex 75. Three PV isoforms—PV‐I, PV‐II, and PV‐III—were obtained and their molecular masses as estimated by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti‐frog PV monoclonal antibody. PV‐I and PV‐II were quite possibly glycoproteins, while PV‐III was not glycosylated, as analyzed by periodic acid–Schiff (PAS) staining. Thermal stability revealed that PV‐I and PV‐II easily formed polymers, while these proteins were stable in a pH range of 4.0–10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. CONCLUSION: Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle. Copyright © 2010 Society of Chemical Industry     

耐热菌D-乳酸脱氢酶突变体的可视化建模和大肠杆菌中突变体对产苯乳酸的影响

以生物信息学为基础,分析D-乳酸脱氢酶（D-LDH）的保守氨基酸残基、活性中心氨基酸残基、蛋白质三维结构和同源建模,可视化比较建模突变体空间构象,优选最佳突变体模型。结果显示,在D-LDH的20个保守氨基酸中,4个与酶活性中心有关。比较突变体模型发现,49和297位的phe或try的苯环形成空间位阻,F49A或Y279A及F49A和Y279A双突变体可解除位阻。对已构建的三个突变体初步发酵显示,IPTG和乳糖都能诱导突变体酶在大肠杆菌中产生苯乳酸,静置培养比摇振培养产量高,用乳糖诱导时,突变体F49A（A.a D-LDH-F49A株）苯乳酸的量比野生型（A.a.D-LDH株）的高。优选可视化突变体可作为高效构建工程菌的方法之一。     

陕西烟区烟草脉带花叶病毒外壳蛋白基因的克隆和原核表达

根据烟草脉带花叶病毒(TVBMV)CP序列合成上下游引物,利用RT-PCR方法获得了(TVBMV)CP基因。序列分析表明,TVBMV CP基因全长813nt,编码271个氨基酸;与GenBank中已报道的9个分离CP基因相比,其核苷酸及氨基酸序列同源性分别为90.28%-98.15%和97.05%-100%。将TVBMV CP基因经BamHI/NotI双酶切定向插入到pET30a载体中,构建了原核表达载体pET30-TVBMV CP,重组质粒经BamHI和NotI双酶切鉴定及基因测序验证正确后,用IPTG进行诱导表达。结果表明:TVBMV CP在大肠杆菌中获得了高效表达,表达产物的分子量为37 kD。用表达产物为抗原免疫家兔制备了特异性抗血清,其效价为1/2048。用Western-blot检测田间样品,其结果表明,制备的抗血清可用于检测田间的发病植株。     

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.     

Purification, immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream, Evynnis japonica

Parvalbumin, a calcium-binding sarcoplasmic protein of 12 kDa, represents a major allergen of fish. Previous immunoblotting experiments suggested the presence of a 14 kDa allergen, together with parvalbumin, in sea breams. In this study, the 14 kDa allergen (PA I) and parvalbumin (PA II) were purified from the white muscle of crimson sea bream, Evynnis japonica, by gel filtration and reverse-phase HPLC. Amino acid sequencing of lysylendopeptidase peptide fragments demonstrated that both PA I and PA II were isoforms of parvalbumin. As analysed by ELISA, PA I showed weaker reactivities with IgG (monoclonal or polyclonal antibody) and serum IgE in fish-allergic patients than did other fish parvalbumins, including PA II. The amino acid sequences of PA I and PA II were elucidated by cDNA cloning. PA I was found to have more specific amino acid residues at several positions compared to the other fish parvalbumins.     

复合保护剂对透明质酸酶的稳定性研究

以球形节杆菌(Arthrobacter globiformis)A152发酵生产的透明质酸酶(HAase)的酶活力为衡量指标,通过研究糖类、醇类、生物大分子和金属离子等保护剂对提高透明质酸酶热稳定性的作用,考察添加复合保护剂对透明质酸酶热稳定性和储存稳定性的影响。结果表明,山梨糖醇、蔗糖和可溶性淀粉作为保护剂,可提高HAase酶的热稳定性。通过单因素和正交实验优化后得到了效果最佳的复合保护剂配方,即山梨醇7.5%、蔗糖10%、可溶性淀粉0.2%。优化后添加保护剂的HAase在42℃保温2 h后,其酶活保留率为88.68%,较对照提高了64.18%,在37℃,通过添加复合保护剂,其半衰期提高了48%。在4℃储藏90 d,添加复合保护剂和防腐剂(0.1%的山梨酸钾和0.05%的苯甲酸钠)的HAase酶活保留率高达85.52%。     

肺炎克雷伯氏杆菌二羟丙酮激酶基因(dhaKL)克隆与表达

以肺炎克雷伯氏菌As1.1736的基因组为模板,通过PCR技术成功地扩增了dhaKL基因,并构建了pET-28a(+)/dhaKL表达载体。DNA序列分析的结果表明:dhaKL基因由2个开放阅读框架组成:ORF1全长为1017bp,编码356个氨基酸;ORF2全长633bp,编码210个氨基酸。SDS-PAGE电泳结果表明:dhaKL基因获得了有效表达,上清液中的酶活性为15.6U/mL。