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81.
The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the
substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic,
cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain
ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side
of the keto group could also be detected yielding the corresponding methyl or ethyl esters. 相似文献
82.
Böttcher D Brüsehaber E Doderer K Bornscheuer UT 《Applied microbiology and biotechnology》2007,73(6):1282-1289
The previously reported functional expression of the γ-isoenzyme of pig liver carboxylesterase (γ-rPLE) in Pichia pastoris is hampered by the small amount of active enzyme formed. Earlier attempts for expression in Escherichia coli failed completely and not even inactive protein was detected. The lack of glycosylation ability of E. coli was ruled out as a possible reason, as it could be shown in this work that deglycosylated PLE also is active. Expression
of γ-rPLE was studied using a range of E. coli strains with careful design of the constructs used and control of the cultivation conditions. Indeed, expression in E. coli strains Rosetta, Origami and Rosetta-gami was successful, but the majority of enzymes was present as inclusion bodies and
only little soluble but inactive protein was detected. Denaturation and refolding of inclusion bodies failed. However, with
the E. coli strain Origami, coexpressing the molecular chaperones GroEL und GroES, a functional expression of γ-rPLE was possible. The
recombinant enzyme was released by cell disruption and subjected to His-tag purification. The purified esterase had a specific
activity of 92 U mg−1 protein and a V
max/K
m value of 10.8×10−3 min−1 towards p-nitrophenyl acetate. Activity staining of native polyacrylamide gels gave a single band at 175 kDa with esterolytic activity
indicating a trimeric form of γ-rPLE (∼60 kDa per monomer). γ-rPLE was biochemically characterized and its properties were
compared to the enzyme previously expressed in P. pastoris. pH and temperature profiles were identical and highest activity was found at pH 8–8.5 and 60 °C, respectively. In the kinetic
resolution of (R,S)-1-phenyl-2-butyl acetate with esterase from both expression hosts, similar enantioselectivities (E=50) were found. 相似文献
83.
Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis
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Mirella Di Lorenzo Aurelio Hidalgo Rafael Molina Juan A. Hermoso Domenico Pirozzi Uwe T. Bornscheuer 《Applied microbiology》2007,73(22):7291-7299
A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme. 相似文献
84.
Marcus Bumgen Theresa Dutschei Daniel Bartosik Christoph Suster Lukas Reisky Nadine Gerlach Christian Stanetty Marko D. Mihovilovic Thomas Schweder Jan-Hendrik Hehemann Uwe T. Bornscheuer 《The Journal of biological chemistry》2021,297(4)
Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides. 相似文献
85.
It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE. 相似文献
86.
U Schmid U T Bornscheuer M M Soumanou G P McNeill R D Schmid 《Biotechnology and bioengineering》1999,64(6):678-684
1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system. 相似文献
87.
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89.
Herter S Nguyen GS Thompson ML Steffen-Munsberg F Schauer F Bornscheuer UT Kourist R 《Applied microbiology and biotechnology》2011,90(3):929-939
The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile
building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two
approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach
was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic
activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative
cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted
tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each
of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing
enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates. 相似文献
90.
The polyol dehydrogenase PDH-11300 from Deinococcus geothermalis was cloned, functionally expressed in Escherichia coli and biochemically characterized. The enzyme showed the highest activity in the oxidation of xylitol and 1,2-hexanediol and had an optimum temperature of 45°C. The enzyme exhibited a T(50)(60)-value of 48.3°C. The T(50)(60) is the temperature where 50% of the initial activity remains after incubation for 1h. In order to elucidate the structural reasons contributing to thermostability, the substrate-binding loop of PDH-11300 was substituted by the loop-region of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scope of this chimera basically represented the average of both wild-type enzymes, but surprisingly the T(50)(60) was noticeably increased by 7°C up to 55.3°C. Further mutations in the active site led to identification of residues crucial for enzyme activity. The cofactor specificity was successfully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD(+) binding cleft, without influencing the catalytic properties of the dehydrogenase. 相似文献