RESUMEN
A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain.After UV-EMS and UV-DES treatments respectively,seven mutated strains with subtle improvements in acid tol-erance were obtained,and were subjected for recursive protoplast fusion.Through three rounds of genome shuffling,four shuffled strains with both higher yield and acid tolerance were obtained.The shuffled strain namely F3-21 could even survive at pH 5.2.The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here.After 48 h of shake-flask fermentation,the succinic acid concentration of F3-21 was 48% higher than that of the parent strain.When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0,the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L,which was increased by 45% compared with that of the parent strain(26.2 g/L).While pH was controlled at 6.5~7.0,the production of succinic acid in 32 h fermentation attained 40.7 g/L.When F3-21 was carried out in fed-batch fermentation,succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation.These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A.succinogenes CGMCC 1593.
RESUMEN
D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.
Asunto(s)
Hidrolasas de Éster Carboxílico , Genética , Barajamiento de ADN , Evolución Molecular Dirigida , Estabilidad de Enzimas , Escherichia coli , Genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes , Genética , Metabolismo , Reacción en Cadena de la Polimerasa , Métodos , Ingeniería de Proteínas , Saccharomyces cerevisiae , GenéticaRESUMEN
The total cDNA obtained through reverse transcription of F. oxysporum CGMCC 0536 mRNA used as template, a fragment about 1.5kb was amplied with oligo(dT)15 primer and a gene specific primer designed on the base of the sequence of both NH2-terminus and the cDNA sequence encoding D-lactonohydrolase of Fusarium oxysporum reported on the NCBI, then the fragment was cloned to the pMD18-T vector and sequenced. The sequence encoding D-lactonohydrolase of F. moniliforme CGMCC 0536 shows a high homology of 90.06% with that of F. oxysporum indicating that the gene encoding D-lactonohydrolase is highly conservative. Two specific primers were designed according to the sequence result, and a fragment, 1146bp, was amplied using hot start PCR with these two specific primers. Subsequently, the resulting products were digested with EcoR I and Sal I and ligated to the pTrc99a vector digested with the same enzymes using T4 DNA ligase. the recombinant plasmid, pTrc99a-LAC, was transformed into Escherichia coli JM109. The two positive clones were induced with IPTG, and enzymes expressed in Escherichia coli JM109, the enzyme activity was about 37U and 41U respectively. The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 40kD protein was obtained.
Asunto(s)
Secuencia de Bases , Hidrolasas de Éster Carboxílico , Genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Genética , Metabolismo , Proteínas Fúngicas , Genética , Fusarium , Genética , Datos de Secuencia MolecularRESUMEN
<p><b>OBJECTIVE</b>To study chemical constituents from pine cone of Pinus annandii.</p><p><b>METHOD</b>The constituents were isolated by chromatographic method and the structures were identified on the basis of spectral analysis.</p><p><b>RESULT</b>Seven compounds were identified as sandaracopimaric acid (I), isodextropimaric acid (II), 12-hydroxyabietic acid (III), dehydroabietic acid (IV), 15-hydroxydehydroabietic acid (V), beta-sitosterol (VI) and daucosterol (VII).</p><p><b>CONCLUSION</b>Compounds I-IV were isolated from this plant for the first time.</p>
Asunto(s)
Diterpenos , Química , Abietanos , Química , Frutas , Química , Pinus , Química , Plantas Medicinales , Química , Sitoesteroles , QuímicaRESUMEN
<p><b>AIM</b>To study chemical constituents from pine cone of Pinus armandii Franch.</p><p><b>METHODS</b>The constituents were isolated by chromatographic method and the structures were identified on the basis of spectral analysis.</p><p><b>RESULTS</b>Four compounds were identified as 7-oxo-12alpha, 13beta-dihydroxyabiet-8(14)-en18-oic acid (I), 7-oxo-13beta-hydroxyabiet-8 (14)-en-18-oic acid (II), 8 (14)-podocarpen-13-on-18-oic acid (III) and lambertianic acid (IV).</p><p><b>CONCLUSION</b>Compound I is a new diterpenoid and compounds II, III were isolated from this plant for the first time.</p>
Asunto(s)
Ácidos Carboxílicos , Química , Diterpenos , Química , Frutas , Química , Conformación Molecular , Naftalenos , Química , Pinus , Química , Plantas Medicinales , QuímicaRESUMEN
The deep fermentation technique of Tricholoma matsutake is systemically studied in this paper firstly. The best culture determined by orthogonal test is 3g/L of cornflour, 1g/L of glucose, 1g/L of bean cake flour, 1mL/L of corn steep liquid, 1g/L of KH 2PO 4. The best fermenting condition is: 25℃, rotating speed 160 r/min, pH5.0,inoculating amount 10%, 120mL culture medium per 500mL flask. Under these conditions, the mycelia reach 12.94g/L after fermenting 12d.
RESUMEN
Using isoeugenol as the sole carbon source,a novel strain,producing high amounts of vanillin from isoeugenol,was isolated from soil.According to the physiological and biochemical characteristics and its 16S rRNA gene sequence analysis,it was identified as Bacillus fusiformis.The initial results showed that 4.20 g/L vanillin was obtained by bioconversion of 2% isoeugenol with Bacillus fusiformis.