Journal of Molecular Catalysis B-enzymatic最新文献

{"title":"Characterization of recombinant β- galactosidase and its use in enzymatic synthesis of lactulose from lactose and fructose","authors":"Xue-Yi Liao ,&nbsp;Qian-Wang Zheng ,&nbsp;Qian-ling Zhou ,&nbsp;Jun-Fang Lin ,&nbsp;Li-Qiong Guo ,&nbsp;Fang Yun","doi":"10.1016/j.molcatb.2016.09.019","DOIUrl":"https://doi.org/10.1016/j.molcatb.2016.09.019","url":null,"abstract":"<div><p>The gene encoding a β- galactosidase was cloned from <em>Lactobacillus plantarum</em> FMNP01 and expressed in <em>Escherichia coli</em> BL21(DE3). The characteristics of this purified recombinant enzyme, L.pFMNP01Gal, were determined, and its transgalactosylation reaction conditions for the production of lactulose were optimized. Using <em>O</em>NPG as substrate, the L.pFMNP01Gal showed specific activity of 980<!--> <!-->U/g with a Km of 6.86<!--> <!-->mM and a Kcat of 22.47/s. This enzyme was most stable at 40–50<!--> <!-->°C, and exhibited optimum catalytic activity at 40<!--> <!-->°C and pH 7.0. The activity of L.pFMNP01Gal was greatly inhibited by Cu²⁺, while other tested metal ions had little influence on it. For the optimization of transgalactosylation reaction, high lactulose production was achieved when 60% (W/V) sugars were used as substrates with a lactose/fructose mass ratio of 2:1, and 2<!--> <!-->U/mL of L.pFMNP01Gal as catalyst. Under these optimum conditions, 18.38<!--> <!-->±<!--> <!-->2.17<!--> <!-->g/L of lactulose was synthesized in 6<!--> <!-->h at 50<!--> <!-->°C. This study provides an alternative method for enzymatic synthesis of lactulose.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 253-260"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.019","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91658175","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conversion of a Monascus ruber esterase into a lipase by disrupting a salt bridge","authors":"Zi-Tong Meng ,&nbsp;Chen Hu ,&nbsp;Yan Zhang ,&nbsp;Hai-Lun Guo ,&nbsp;Mu Li","doi":"10.1016/j.molcatb.2016.11.012","DOIUrl":"10.1016/j.molcatb.2016.11.012","url":null,"abstract":"<div><p>Cold-active lipases have emerged as an important class of biocatalysts for chemical and food industries due to their high efficiency at low temperature and long-chain substrate preference. In an effort to explore the feasibility of converting a cold-active esterase from <em>Monascus ruber</em> (Lip10) into a cold-active lipase, an Y264F variant in which the salt bridge between K243 and Y264 was disrupted has been constructed and characterized. The interfacial kinetic parameter, K_m^app for pNP-laurate (C12) and pNP-palmitate (C16), of Lip10 esterase was 4.2 and 5.7 times higher than those of the Y264F variant, respectively. Substrate specificity of the Y264F variant changed from shot-chain length substrate to medium- and long-chain length substrates, indicating that the Y264F variant turned into a lipase. Meanwhile, the Y264F variant displayed 48.6% maximum activity at 4<!--> <!-->°C and 3.2<!--> <!-->kcal/mol activation energy in the range of 5–30<!--> <!-->°C, suggesting that it was still cold-active. Based on analysis of the structure-function relationships, it suggests that the shape of substrate channel controlled by the conserved salt bridge was very important for the substrate specificity. This study provides a way to alter the substrate preference of the Lip10 esterase as well as new insight into the structural basis of esterase substrate specificity.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 178-185"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.11.012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79425832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression and characterization of a thermostable organic solvent-tolerant laccase from Bacillus licheniformis ATCC 9945a","authors":"Nikola Lončar ,&nbsp;Nataša Božić ,&nbsp;Zoran Vujčić","doi":"10.1016/j.molcatb.2016.06.005","DOIUrl":"10.1016/j.molcatb.2016.06.005","url":null,"abstract":"<div><p>Bacterial laccases have proven advantages over fungal and plant counterparts in terms of wider pH optimum, higher stability and broader biocatalytic scope. In this work, <em>Bacillus licheniformis</em> ATCC 9945a laccase is produced heterologously in <em>Escherichia coli</em>. Produced laccase exhibits remarkably high temperature optimum at 90<!--> <!-->°C and possess significant thermostability and resistance to inactivation by organic solvents. Laccase has an apparent melting temperature of 79<!--> <!-->°C at pH 7.0 and above 70<!--> <!-->°C in range of pH 5.0–8.0, while having half-life of 50<!--> <!-->min at 70<!--> <!-->°C. Presence of 10% organic solvents such as acetonitrile, dimethylformamide, dimethylsulfoxide or methanol reduces melting temperature to 45–52<!--> <!-->°C but activity remains practically unimpaired. With 50% of acetonitrile and methanol laccase retained ∼40% of initial activity. EDTA and 300<!--> <!-->mM sodium-chloride have positive effect on activity. Enzyme is active on syringaldazine, ABTS, phenols, amines, naphthol, lignin and lignin model compounds and mediates C<img>C bond formation <em>via</em> oxidative coupling after one electron oxidation of phenolic group. Successful polymerization of 2-naphthol was achieved with 77% conversion of 250<!--> <!-->mg/L 2-naphtol in only 15<!--> <!-->min which may further expand substrate scope of this enzyme towards polymer production and/or xenobiotics removal for environmental applications.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 390-395"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.06.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83472527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evolved alkaline fungal laccase secreted by Saccharomyces cerevisiae as useful tool for the synthesis of C–N heteropolymeric dye","authors":"Ana I. Vicente ,&nbsp;Javier Viña-Gonzalez ,&nbsp;Paloma Santos-Moriano ,&nbsp;Carlos Marquez-Alvarez ,&nbsp;Antonio O. Ballesteros ,&nbsp;Miguel Alcalde","doi":"10.1016/j.molcatb.2016.10.004","DOIUrl":"10.1016/j.molcatb.2016.10.004","url":null,"abstract":"<div><p>Enzymatic production of C–N heteropolymeric dyes at alkaline pHs is an attractive process for the textile industry. In this work, we have designed a fungal laccase by directed evolution so that it may be used at alkaline pHs for the synthesis of C–N heteropolymeric dyes (C–N polydye) from catechol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA). Firstly, several medium- and high-redox potential fungal laccases from previous laboratory evolution campaigns were benchmarked for the synthesis of the C–N polydye at pH 8.0, choosing an alkaline laccase mutant from <em>Myceliophthora thermophila</em> as the departure point for further engineering. Mutant libraries were then constructed, expressed in <em>Saccharomyces cerevisiae</em> and screened using a high-throughput colorimetric assay for the detection of the C–N polydye. By combining directed and focused molecular evolution, a novel, strongly expressed alkaline laccase variant was identified. This laccase was secreted at 37<!--> <!-->mg/L and its catalytic efficiency for the oxidation of catechol and 2,5-DABSA at pH 8.0 was enhanced 3.5-fold relative to that of the wild-type, promoting the synthesis of the C–N polydye at basic pHs. While the improved expression was mostly the result of accumulating mutations that favor the yeast’s codon usage together with the recovery of a secretion mutation, the enhanced C–N polydye synthetic activity of the mutant laccase was dependent on the alkaline mutations it inherited. Readily secreted, this laccase mutant would appear to be a valuable platform for organic synthesis at basic pHs.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 323-330"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90380196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX","authors":"Dana I. Colpa,&nbsp;Marco W. Fraaije","doi":"10.1016/j.molcatb.2016.08.017","DOIUrl":"10.1016/j.molcatb.2016.08.017","url":null,"abstract":"<div><p>The heterologous overexpression level of the bacterial dye decolorizing peroxidase <em>Tfu</em>DyP in <em>Escherichia coli</em> was increased sixty fold to approximately 200<!--> <!-->mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly overexpressed SUMO-<em>Tfu</em>DyP was, however, almost inactive. Analysis of the enzyme by UV–vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly overexpressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 372-377"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.08.017","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85911898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification and characterization of an alkaline chloride-tolerant laccase from a halotolerant bacterium, Bacillus sp. strain WT","authors":"Maryam Siroosi ,&nbsp;Mohammad Ali Amoozegar ,&nbsp;Khosro Khajeh","doi":"10.1016/j.molcatb.2016.10.001","DOIUrl":"10.1016/j.molcatb.2016.10.001","url":null,"abstract":"<div><p>Laccases are multicopper oxidases with various biotechnological applications that oxidize different aromatic or inorganic substrates. In present work, different bacterial strains isolated from Urmia lake, a hypersaline lake in northwest of Iran, were screened to find laccase-producing ones. Spore and an extracellular enzyme from a halotolerant spore-forming bacterium<em>, Bacillus</em> sp. strain WT, showed laccase activity toward typical laccase substrates: syringaldazine and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate). The extracellular laccase (0.01<!--> <!-->U<!--> <!-->mL⁻¹) decolorized sulphonyl green BLE up to 97% at pH 7.0 after two h incubation at 35<!--> <!-->°C, without any addition of mediators. This enzyme with apparent molecular mass of 180<!--> <!-->kDa was purified using ammonium sulfate precipitation method and anion exchange chromatography. The optimum laccase activity toward 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine was at 55<!--> <!-->°C and pH values of 5.0 and 8.0, respectively. One mM of metal ions, Na⁺ and Ni²⁺, increased the enzyme activity by 12%. The enzyme from <em>Bacillus</em> sp. strain WT could be able to tolerate up to 600–800<!--> <!-->mM NaCl (a very strong laccase inhibitor) and showed halotolerant nature with maximum activity at 100<!--> <!-->mM NaCl. One mM NaN₃ (another potent laccase inhibitor) almost had no effect on the laccase activity; however, 1<!--> <!-->mM <span>l</span>-Cys reduced 87% of its original activity. <em>K</em>_M values for the purified enzyme on 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine were determined to be 132.7 and 3.7<!--> <!-->μM, with corresponding <em>k</em>_cat values of 309 and 51<!--> <!-->s⁻¹, respectively. The present study is among the first studies on laccase activity of a halotolerant bacterial strain isolated from a hypersaline lake.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 89-97"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84466890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation on the underlying mechanism: How fusion xylanase-ELPs self-assembles into insoluble active aggregates","authors":"Shuyu Wu ,&nbsp;Yilin Wang ,&nbsp;Tingting Shen ,&nbsp;Junhui Wang ,&nbsp;Guangya Zhang","doi":"10.1016/j.molcatb.2016.10.012","DOIUrl":"10.1016/j.molcatb.2016.10.012","url":null,"abstract":"<div><p>We have successfully obtained a recombinant xylanase by fusing with elastin-like polypeptides (ELPs), the xylanase SoxB underwent a sharp irreversible phase transition, and self-assembled into an insoluble but more catalytically operative particle. This was analogous to the immobilized xylanase to a large extent and aroused our interest to gain new insights into the determinant factor that may cause this phenomenon. We herein listed several candidate factors including the length of ELPs, linker sequence, buffer properties, and the target protein, subsequently we evaluated their contributions to the formation of the active aggregates. The results suggested that SoxB was fused with ELPs as desired protein partners, neither ELPs length nor the linker type made crucial contribution to the formation of active aggregates. However, when Na₂CO₃ was chosen as the salt to trigger the phase transition, the catalytic activities detected in aggregates accounted for more than 87.7% of total activity, whereas above 83.8% of the activity remained in supernatant when using Na₂SO₄. Then we introduced an alkali-tolerant xylanase termed as Xyl and compared it with SoxB, and found that the activity ratio in insoluble particle dropped to 15.3% in Na₂CO₃ and 19.3% in Na₂SO₄ respectively, only a few insoluble aggregates observed during the purification. Therefore, we speculated the property of xylanase partner fused to ELPs should be the predominant factor to form the catalytically active insoluble aggregates, and this provides a promising not yet reported perspective for industrial application of “immobilized” SoxB.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 247-252"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86336573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"One-pot synthesis of nitrocyclopropane: α-Amylase-catalyzed Michael addition initiated ring-closure sequence reactions","authors":"Xue-Dong Zhang,&nbsp;Jian Song,&nbsp;Na Gao,&nbsp;Zhi Guan,&nbsp;Yan-Hong He","doi":"10.1016/j.molcatb.2016.09.006","DOIUrl":"10.1016/j.molcatb.2016.09.006","url":null,"abstract":"<div><p>This article presents a one-pot synthesis of nitrocyclopropanes via Michael addition initiated ring-closure sequence reactions of bromonitroalkane to α,β-unsaturated enones. Moderate to favorable yields (55–93%) and certain enantioselectivities were obtained with α-amylase from hog pancreas as the catalyst. This strategy utilizes the unnatural ability of enzymes to provide a convenient and biocatalytic method for green organic synthesis.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 1-8"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81801920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemodivergent fungal oxidation of isochroman","authors":"Gabriela I. Furque ,&nbsp;Fabricio R. Bisogno ,&nbsp;Virginia E. Sosa","doi":"10.1016/j.molcatb.2016.10.015","DOIUrl":"10.1016/j.molcatb.2016.10.015","url":null,"abstract":"<div><p>This work extends the present knowledge about the ability of filamentous fungi and Baker’s Yeast to selectively transform oxygen-containing compounds. Previously, it has been demonstrated that several species of the <em>Aspergillus</em> genus are able to perform selective oxidation of benzopyrans. Isochroman or 3,4-dihydro-1<em>H</em>-benzopyran (<strong>1</strong>) was chosen as model substrate for the biotransformation since related motifs are often found in the structure of natural products with important biological and pharmacological activities. All the tested strains showed the ability to oxidize <strong>1</strong>. Chemodivergent reaction pathways between the employed microorganisms were observed. The use of cytochrome P450 enzyme inhibitors, and different oxygenation conditions allowed to inquire about the type of enzymes involved in the process. The results obtained were compared with chemical one-electron oxidation of compound <strong>1</strong> and thus, a metabolic pathway was proposed.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 310-316"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.015","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86899235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic modification and optimization of endo-inulinase for the enzymatic production of oligofructose from inulin","authors":"Jiang-Ke Yang,&nbsp;Ji-Wen Zhang,&nbsp;Lin Mao,&nbsp;Xun You,&nbsp;Guang-Jun Chen","doi":"10.1016/j.molcatb.2016.10.020","DOIUrl":"10.1016/j.molcatb.2016.10.020","url":null,"abstract":"<div><p>The enzymatic hydrolyzation of inulin by endo-inulinase to produce oligofructoses, a new type of food additive and health product, is a promising, “green”, and environmentally friendly technique. To identify novel genetic sources of endo-inulinase genes and facilitate their industrial application for oligofructose production, we cloned an endo-inulinase gene from a <em>Fusarium oxysporum</em> strain and achieved high-level expression in the genetically modified <em>Pichia pastoris</em><span> strain in a pilot-scale bioreactor by using strategies such as C-terminal truncation and mutagenesis of protease-sensitive sites. We then optimized the parameters of the inulinase reaction and the amount of enzyme used to inulin hydrolysis and oligofructose production. The results of this study should facilitate the bulk production of inulinase and provide a reference for the industrial production of oligofructose from inulin.</span></p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 225-232"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78665551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}