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1.
2.
The aim of this work has been to study the substrate specificity of two aromatic peroxygenases concerning polyaromatic compounds
of different size and structure as well as to identify the key metabolites of their oxidation. Thus, we report here on new
pathways and reactions for 2-methylnaphthalene, 1-methylnaphthalene, dibenzofuran, fluorene, phenanthrene, anthracene and
pyrene catalyzed by peroxygenases from Agrocybe aegerita and Coprinellus radians (abbreviated as AaP and CrP). AaP hydroxylated the aromatic rings of all substrates tested at different positions, whereas
CrP showed a limited capacity for aromatic ring-hydroxylation and did not hydroxylate phenanthrene but preferably oxygenated
fluorene at the non-aromatic C9-carbon and methylnaphthalenes at the side chain. The results demonstrate for the first time the broad substrate specificity
of fungal peroxygenases for polyaromatic compounds, and they are discussed in terms of their biocatalytic and environmental
implications. 相似文献
3.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
4.
Kluge M Ullrich R Dolge C Scheibner K Hofrichter M 《Applied microbiology and biotechnology》2009,81(6):1071-1076
Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene.
Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144
and a characteristic UV absorption spectrum (A
max 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into
1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene
1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using 18O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts
both as oxidant (primary electron acceptor) and oxygen source. 相似文献
5.
Nguyen VN Oh IJ Kim YJ Kim KY Kim YC Park RD 《Journal of industrial microbiology & biotechnology》2009,36(2):195-203
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography.
The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and
showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino
acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46
were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag+ and Hg2+ while Chi46 by Hg2+ and Pb2+ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co2+. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)
n
, n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase. 相似文献
6.
Berbert-Molina MA Prata AM Pessanha LG Silveira MM 《Journal of industrial microbiology & biotechnology》2008,35(11):1397-1404
The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S
0) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically
well-defined culture phases, independent of S
0 values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation
and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ
X,m) were not affected with S
0 up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by
a reduction in μ
X,m values. These higher S
0 values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X
m = 15.2–15.9 g/l) were achieved with S
0 over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose
to form other metabolites. In this case, with S
0 from 30.8 to 152 g/l, cell yield (Y
X/S
) decreased from 0.58 to 0.41 g/g. On the other hand, with S
0 = 10 g/l growth was limited by substrate, and Y
X/S
has shown its maximum value (0.83 g/g). 相似文献
7.
Shockey J Chapital D Gidda S Mason C Davis G Klasson KT Cao H Mullen R Dyer J 《Applied microbiology and biotechnology》2011,92(6):1207-1217
Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has
not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable
to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly
upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular
fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular
lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells
to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular
lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion
of low-value lipids to value-added TAGs and other novel lipid products. 相似文献
8.
Wei Heng Jun Wu Huaqing Wu Yufen Cao Takeshi Nishio Shao-Ling Zhang 《Molecular breeding : new strategies in plant improvement》2011,28(4):549-557
Pyrus displays gametophytic self-incompatibility controlled by a single highly polymorphic gene complex termed S locus, which comprises a stylar-expressed gene (S-RNase) tighlty linked with a pollen expressed gene, that determines the specificity of the self-incompatibility locus. Deduced
amino acid sequence of ‘Meigetsu’ S
8
-RNase in Pyrus pyrifolia and ‘Kuerlexiangli’ S
28
-RNase in P. sinkiangensis showed 100% identity. S
3
-RNase in Malus
spectabilis was also found to be similar to S
8
-RNase in P. pyrifolia with 96.9% identity in the deduced amino acid sequence. The intron, which is generally highly polymorphic between alleles,
was also remarkably well conserved within these allele pairs. The intron of PpS
8
-RNase showed 95.3 and 91.9% identity with PsS
28
-RNase and MsS
3
-RNase, respectively. Pollen tube growth in styles, pollen tube length in artificial media containing different S-RNases and
segregation of S haplotypes in F1 plants revealed commonality of the recognition specificity between PpS
8
-RNase and PsS
28
-RNase and between PpS
8
-RNase and MsS
3
-RNase. Results suggested that PpS
8
-RNase, PsS
28
-RNase and MsS
3
-RNase have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions
found between PpS
8
-RNase and MsS
3
-RNase do not alter the recognition specificity. 相似文献
9.
Talita A. Sampaio e Silva Adriana Knob Célia R. Tremacoldi Marcia R. Brochetto-Braga Eleonora Cano Carmona 《World journal of microbiology & biotechnology》2011,27(11):2491-2497
Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most
of these applications the enzymatic preparation must be at least partially purified and free of substances that could change
the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained
mainly from Mucor, Rhizopus, Penicillium
and
Aspergillus species. A strain of Aspergillus
clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was
purified 37.2 times with 37% recovery using (NH4)2SO4 fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa.
The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH
stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg+2 and partially inhibited by Cu+2, Zn+2 and Mn+2. The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The
protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited
by pepstatin, with a Ki of 7.8 μM, indicating that is an aspartic protease. A.
clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and
dietary supplements, in which activity and stability in acid pH are required. 相似文献
10.
Christos P. Papaneophytou Anastasia A. Pantazaki Dimitrios A. Kyriakidis 《Applied microbiology and biotechnology》2009,83(4):659-668
The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear
zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. The purified enzyme
was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the
TTHA0199 gene product of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other carboxylesterases. A catalytic
triad was identified consisting of S183, E310, and H405. A pentapeptide sequence (GX1SX2G) exists within the molecule, characteristic for PHB depolymerases (lipase box) and for other serine hydrolases. Purified
extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of
the purified enzyme for PHB was 53 μg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate
was identified, suggesting that the enzyme acts principally as an exo-type hydrolase. 相似文献
11.
We previously identified both self-incompatible and self-compatible plants in a natural population of self-incompatible Petunia axillaris subsp. axillaris, and found that all the self-compatible plants studied carried either SC1- or SC2-haplotype. Genetic crosses showed that SC2 was identical to S17 identified from another natural population of P. axillaris, except that its pollen function was defective, and that the pollen-part mutation in SC2 was tightly linked to the S-locus. Recent identification of the S-locus F-box gene (SLF) as the gene that controls pollen specificity in S-RNase-based self-incompatibility has prompted us to examine the molecular basis of this pollen-part mutation. We cloned and sequenced the S17-allele of SLF of P.axillaris, named PaSLF17, and found that SC2 SC2 plants contained extra restriction fragments that hybridized to PaSLF17 in addition to all of those observed in S17 S17 plants. Moreover, these additional fragments co-segregated with SC2. We used the SC2-specific restriction fragments as templates to clone an allele of PaSLF by PCR. To determine the identity of this allele, named PaSLFx, primers based on its sequence were used to amplify PaSLFalleles from genomic DNA of 40 S-homozygotes of P. axillaris, S1 S1 through S40 S40. Sequence comparison revealed that PaSLFx was completely identical with PaSLF19 obtained from S19 S19. We conclude that the S-locus of SC2 contained both S17-allele and the duplicated S19-allele of PaSLF. SC2 is the first naturally occurring pollen-part mutation of a solanaceous species that was shown to be associated with duplication of the pollen S. This finding lends support to the proposal, based on studies of irradiation-generated pollen-part mutants of solanaceous species, that duplication, but not deletion, of the pollen S, causes breakdown of pollen function. 相似文献
12.
Katsuyuki Kakeda 《Plant cell reports》2009,28(9):1453-1460
Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled
by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification
and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum
S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S
3 haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed
that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from
S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in
the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S
3) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although
the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three
alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825
and AB511859–AB511862. 相似文献
13.
Hai-Nan Su Bin-Bin Xie Xiu-Lan Chen Jin-Xia Wang Xi-Ying Zhang Bai-Cheng Zhou Yu-Zhong Zhang 《Journal of applied phycology》2010,22(1):65-70
Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple
and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding
hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed
by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude
phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0).
In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A
650/A
280 of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g−1 wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from
5.6 to 4.0. The absorbance ratio A
650/A
280 of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g−1 wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. 相似文献
14.
Eriko Oikawa Shohei Takuno Atsushi Izumita Koji Sakamoto Hideaki Hanzawa Hiroyasu Kitashiba Takeshi Nishio 《Molecular breeding : new strategies in plant improvement》2011,28(1):1-12
In F1 hybrid breeding of Brassica vegetables utilizing the self-incompatibility system, identification of S genotypes in breeding lines is required. In the present study, we developed S-tester lines of 87 S haplotypes, i.e., 42 S haplotypes in B. rapa and 45 S haplotypes in B. oleracea. With these materials, we established a simple, efficient, and reliable dot-blot technique for S genotyping for 40 S haplotypes of B. rapa and and 33 of B. oleracea using allele-specific oligonucleotide probes and allele-specific primer pairs designed from sequences of each SP11 allele. In this method, DNA fragments amplified using multiplex primer pairs with digoxigenin-dUTP were hybridized with dot-blotted
allele-specific oligonucleotide probes with distinct signals. In addition, we developed a screening method for identification
of plants harboring a particular S haplotype using a labeled allele-specific oligonucleotide probe. This method is considered to be useful for purity testing
of F1 hybrid seeds. 相似文献
15.
A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S2, very similar to S2-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S2 also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S1S5 and S2S4, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S2-specific primers, TAC clones containing both AhSLF-S2 and its homologs were subsequently identified (S2TAC, S5TACa, S4TAC, and S1TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S2, -S1, -S4 and -S5) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S2 were identified. In total, three F-box genes (AhSLF-S2, -S2A and -S2C) in S2TAC (51 kb), three (AhSLF-S4, -S4A and -S4D) in S4TAC (75 kb), two (AhSLF-S5 and -S5A) in S5TACa (55 kb), and two (AhSLF-S1 and -S1E) in S1TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S2C, -S4D and -S1E) were specifically expressed in pollen, similar to AhSLF-S2, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535 相似文献
16.
A. BarbosaJr L. H. S. GuimarÃes H. F. Terenzi J. A. Jorge F. A. Leone M. L. T. M. Polizeli 《Folia microbiologica》2008,53(6):509-516
The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1–2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular
AlPs were purified 5.0 and 9.3×, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and
120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for
both forms. Mn2+, Na+ and Mg2+ stimulated the activity, while Al3+ and Zn2+ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 °C and pH 8.0, respectively.
The enzymes were stable at 50 °C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K
m 0.28 and 0.22 mmol/L, with υ
lim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable
for biotechnological application. 相似文献
17.
Maofu Li Kaikai Zhu Songling Bai Zhi Liu Tianzhong Li 《Molecular breeding : new strategies in plant improvement》2011,28(2):171-180
Apple (Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB (S-RNase-linked F-box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others
appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB-like genes, which showed pollen-specific expression without S-allelic polymorphism. 相似文献
18.
Cytochrome c
6
, (cyt c
6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration
column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa
and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure
as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E
m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron
transfer processes is discussed. 相似文献
19.
Jian-Dong Zhang Ai-Tao Li Hui-Lei Yu Tadayuki Imanaka Jian-He Xu 《Journal of industrial microbiology & biotechnology》2011,38(5):633-641
A cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. can catalyze asymmetric oxygenation of sulfides to S-sulfoxides. However, P450SMO-catalyzed biotransformations require a constant supply of NAD(P)H, the expense of which constitutes
a great hindrance for this enzyme application. In this study, we investigated the asymmetric oxygenation of sulfide to S-sulfoxide using E. coli cells, which co-express both the P450SMO gene from Rhodococcus sp. and the glucose dehydrogenase (GDH) gene from Bacillus subtilis, as a catalyst. The results showed that the catalytic performance of co-expression systems was markedly improved compared
to the system lacking GDH. When using recombinant E. coli BL21 (pET28a-P450-GDH) whole cell as a biocatalyst, NADPH was efficiently regenerated when glucose was supplemented in the
reaction system. A total conversion of 100% was achieved within 12 h with 2 mM p-chlorothioanisole substrate, affording 317.3 mg/L S-sulfoxide obtained. When the initial sulfide concentration was increased to 5 mM, the substrate conversion was also increased
nearly fivefold: S-sulfoxide amounted to 2.5 mM (396.6 mg/L) and the ee value of sulfoxide product exceeded 98%. In this system, the effects of glucose concentration and substrate concentration
were further investigated for efficient biotransformation. This system is highly advantageous for the synthesis of optically
pure S-sulfoxide. 相似文献
20.
The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1–6) are composed of two steps: (1) formation of the non-covalent enzyme–inhibitor complex (E:I) from the inhibitor and the enzyme
and (2) formation of the tetrahedral enzyme–inhibitor adduct (E–I) from the E:I complex. From a stopped-flow apparatus, the
dissociation constant for the E:I complex, KS, and the rate constant for formation of the tetrahedral E–I adduct from the E:I complex, k2 are obtained from the non-linear least-squares of curve fittings of first-order rate constant (kobs) versus inhibition concentration ([I]) plot against kobs=k2+k2[I]/(KS+[I]). Values of pKS, and log k2 are linearly correlated with the σ* values with the ρ* values of −2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to
the ρ* value of −2.0. The tetrahedral E–I adducts on the other hand are more negative charges than the E:I complexes due to the
ρ* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the
pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme. 相似文献