Identification of key sites determining the cofactor specificity and improvement of catalytic activity of a steroid 5β-reductase from Capsella rubella.


:Progesterone 5β-reductases (P5βRs) are involved in 5β-cardenolide formation by stereo-specific reduction of the △4,5 double bond of steroid precursors. In this study a steroid 5β-reductase was identified in Capsella rubella (CrSt5βR1) and its function in steroid 5β-reduction was validated experimentally. CrSt5βR1 is capable of enantioselectively reducing the activated CC bond of broad substrates such as steroids and enones by using NADPH as a cofactor and therefore has the potential as a biocatalyst in organic synthesis. However, for industrial purposes the cheaper NADH is the preferred cofactor. By applying rational design based on literature and complementary mutagenesis strategies, we successfully identified two key amino acid residues determining the cofactor specificity of the enzyme. The R63 K mutation enables the enzyme to convert progesterone to 5β-pregnane-3,20-dione with NADH as cofactor, whereas the wild-type CrSt5βR1 is strictly NADPH-dependent. By further introducing the R64H mutation, the double mutant R63K_R64H of CrSt5βR1 was shown to increase enzymatic activity by13.8-fold with NADH as a cofactor and to increase the NADH/NADPH conversion ratio by 10.9-fold over the R63 K single mutant. This finding was successfully applied to change the cofactor specificity and to improve activity of other members of the same enzyme family, AtP5βR and DlP5βR. CrSt5βR1 mutants are expected to have the potential for biotechnological applications in combination with the well-established NADH regeneration systems.


Enzyme Microb Technol


Li Y,Pan H,Chang Y,Dong N,Zou L,Liang P,Tian W,Chang Z




Has Abstract


2020-03-01 00:00:00












  • The development and application of a new affinity partitioning system for enzyme isolation and purification.

    abstract::A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a str...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Nguyen AL,Luong JH

    更新日期:1990-09-01 00:00:00

  • Identification, repair and characterization of a benzyl alcohol-inducible promoter for recombinant proteins overexpression in Corynebacterium glutamicum.

    abstract::Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Liu X,Zhao Z,Dong G,Li Y,Peng F,Liu C,Zhang F,Linhardt RJ,Yang Y,Bai Z

    更新日期:2020-11-01 00:00:00

  • Porous biochar/chitosan composites for high performance cellulase immobilization by glutaraldehyde.

    abstract::In this work, porous biochar derived from sugarcane bagasse was prepared and then coated with different amounts of chitosan (C@CS) for cellulase immobilization. Cellulase was covalently immobilized on the support by using glutaraldehyde as a linker. The chemical characteristics and morphology of the samples were deter...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Mo H,Qiu J,Yang C,Zang L,Sakai E,Chen J

    更新日期:2020-08-01 00:00:00

  • Esterases of xylan-degrading microorganisms: production, properties, and significance.

    abstract::This review focuses on the description of recently discovered esterase enzymes involved in xylan degradation (acetyl xylan, feruloyl, and p-coumaroyl esterases). The occurrence of these enzymes in various microorganisms, assays used for determination of their activity, induction and production on different substrates,...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章,评审


    authors: Christov LP,Prior BA

    更新日期:1993-06-01 00:00:00

  • Screening of whole-cell immobilization procedures for the delta 1-dehydrogenation of steroids in organic medium.

    abstract::Different whole-cell immobilization methods and conditions were tested for the delta 1-dehydrogenation of 6-alpha-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could no...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Pinheiro HM,Cabral JM

    更新日期:1992-08-01 00:00:00

  • Studies on the purification of peroxidase from horseradish roots using reverse micelles.

    abstract::Horseradish peroxidase (HRP) was successfully purified from horseradish roots by a two-stage reverse-micellar extraction from the dialyzed aqueous extract. The anionic surfactant AOT dissolved in isooctane was used to produce the reverse-micellar phases. The narrow pH range at which HRP solubilization occurred was exp...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Regalado C,Asenjo JA,Pyle DL

    更新日期:1996-04-01 00:00:00

  • Membrane filtration biocathode microbial fuel cell for nitrogen removal and electricity generation.

    abstract::Conductive materials with attached biofilms, were used as membrane filtration biocathodes to filter the effluent and supply electrons for denitrification. Stainless steel mesh and carbon felt were employed to fabricate membrane modules, and the two MFC systems were termed as M1 and M2, respectively. High effluent qual...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Zhang G,Zhang H,Ma Y,Yuan G,Yang F,Zhang R

    更新日期:2014-06-10 00:00:00

  • Comments on "Bioprocessing in space"

    abstract::An analysis developed by Westgate et al. for the digestible energy of edible and inedible biomass, including hydrolysis and fermentation, is reexamined with state-of-the-art values for the harvest index of hydroponic crops. ...

    journal_title:Enzyme and microbial technology

    pub_type: 评论,杂志文章


    authors: Volk T

    更新日期:1993-10-01 00:00:00

  • Characterization of specificity of subtilisin Carlsberg towards peptide T by high-performance liquid chromatography and electrospray mass spectrometry.

    abstract::Peptide T has a sequence (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) belonging to HIV envelope that is involved in the interaction with CD(4) receptor of T lymphocytes. Research of protease activities towards this peptide is very relevant for AIDS therapy. Characterization of specificity of subtilisin Carlsberg towards this ver...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Nedjar-Arroumea N,Paon M,Koralewski F,Friboulet A,Kouach M,Briand G,Leroy Y,Guillochon D

    更新日期:2000-03-01 00:00:00

  • Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases.

    abstract::The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Bonnina E,Brunel M,Gouy Y,Lesage-Meessen L,Asther M,Thibault J

    更新日期:2001-01-02 00:00:00

  • A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10.

    abstract::beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC and xylose isomerase (D-xylose ketol-isomerase EC produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xyl...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Belfaquih N,Penninckx MJ

    更新日期:2000-07-01 00:00:00

  • Cloning and characterization of a novel phenylalanine ammonia-lyase gene from Inonotus baumii.

    abstract::Phenylalanine ammonia-lyase (PAL) gene plays an important role in the synthesis of flavones, lignin, and other bioactive compounds in living organisms. Inonotus baumii, the only known flavone-producing filamentous fungus, is of great importance in the investigation of flavone metabolic pathways. To study the function ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Lin W,Liu A,Weng C,Li H,Sun S,Song A,Zhu H

    更新日期:2018-05-01 00:00:00

  • Transient behavior of T-cell hybridomas in response to changes in metabolite concentrations in continuous culture.

    abstract::Effects of pulse and step changes in major metabolite concentrations such as glucose, glutamine, lactic acid, and ammonium on behavior of T-cell hybridomas in continuous culture were investigated in order to develop a strategy to maximize specific rate of lymphokine formation. Step increases in glucose, lactic acid, a...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Shaughnessy TS,Kargi F

    更新日期:1990-09-01 00:00:00

  • Intracellular synthesis of gold nanoparticles with antioxidant activity by probiotic Lactobacillus kimchicus DCY51T isolated from Korean kimchi.

    abstract::A straightforward synthesis of gold nanoparticles (AuNps) is achieved by novel probiotic Lactobacillus kimchicus DCY51T isolated from Korean kimchi via an intracellular membrane-bound mechanism. The bioreduction of HAuCl4 into AuNps was verified by ultraviolet-visible spectrophotometry at ∼540nm. AuNps were spherical ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Markus J,Mathiyalagan R,Kim YJ,Abbai R,Singh P,Ahn S,Perez ZEJ,Hurh J,Yang DC

    更新日期:2016-12-01 00:00:00

  • Catalytic efficiency of a multi-domain transglycosylating chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) is influenced by polycystic kidney disease domains.

    abstract::Bacterial chitinases recruited multiple accessory domains for the conversion of recalcitrant polysaccharides to simple soluble sugars/amino sugars. Here, we report detailed properties of a multi-domain GH18 chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) that preferred β-chitin as substrate. EcChi2 exhibit...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Mallakuntla MK,Podile AR

    更新日期:2021-02-01 00:00:00

  • Chain length selectivity during the polycondensation of siloxane-containing esters and alcohols by immobilized Candida antarctica lipase B.

    abstract::We have examined the chain length selectivity for a series of acyl donors by lipase B from Candida antarctica (CalB). CalB accepted aliphatic diesters of C4, C6 and C12 chain lengths equally. The introduction of a carbon-carbon double bond into the C4 esters dramatically lowered the rate constant associated with polym...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Frampton MB,Zelisko PM

    更新日期:2014-05-10 00:00:00

  • Stabilization of Penicillium occitanis cellulases by spray drying in presence of Maltodextrin.

    abstract::The stabilization of fungal cellulases by spray drying, the thermal stability of Penicillium occitanis cellulases and the effect of some additives were studied. We observed that CMCase activity presents a good stability at 50 degrees C, even after 60 h of incubation. On the other hand, beta-glucosidase activity was mo...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Belghith H,Ellouz Chaabouni S,Gargouri A

    更新日期:2001-02-01 00:00:00

  • Enzymatic production of cephalexin.

    abstract::Enzymatic production of cephalexin using immobilized penicillin G acylase was studied. The factors that affect the conversion of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) to cephalexin, such as pH; temperature; concentrations of 7-ADCA, D(-)-phenylglycine methyl ester (PGME), and immobilized penicillin G acylas...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Maladkar NK

    更新日期:1994-08-01 00:00:00

  • Modulatory effect of low-shear modeled microgravity on stress resistance, membrane lipid composition, virulence, and relevant gene expression in the food-borne pathogen Listeria monocytogenes.

    abstract::The present study investigated the influence of low-shear modeled microgravity (LSMMG) conditions on Listeria monocytogenes stress response (heat, cold, and acid), membrane fatty acid composition, and virulence potential as well as stress-/virulence-associated gene expression. The results showed that LSMMG-cultivated ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Sheet S,Yesupatham S,Ghosh K,Choi MS,Shim KS,Lee YS

    更新日期:2020-02-01 00:00:00

  • Magnetosomes extracted from Magnetospirillum magneticum strain AMB-1 showed enhanced peroxidase-like activity under visible-light irradiation.

    abstract::Magnetosomes are intracellular structures produced by magnetotactic bacteria and are magnetic nanoparticles surrounded by a lipid bilayer membrane. Magnetosomes reportedly possess intrinsic enzyme mimetic activity similar to that found in horseradish peroxidase (HRP) and can scavenge reactive oxygen species depending ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Li K,Chen C,Chen C,Wang Y,Wei Z,Pan W,Song T

    更新日期:2015-05-01 00:00:00

  • Chaperon solvent plug design in size-exclusion chromatography protein refolding process.

    abstract::Although the chaperon solvent plug was reported as a strategy to reduce aggregation before the column inlet in SEC (size-exclusion chromatography) protein refolding process, the appropriate position at which sample injected and the volume of the chaperon solvent plug have not been elucidated. Therefore, the detail of ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Chen YC,Liu HS

    更新日期:2011-07-10 00:00:00

  • New-to-nature sophorose analog: a potent inducer for gene expression in Trichoderma reesei.

    abstract::Controlled hydrolysis of lactonic sophorolipids from Starmerella bombicola yields a previously undescribed sophorose analog that potently induces cellulase in Trichoderma reesei Rut-C30. Acid treatment of natural sophorolipids results in a mixture of monoacetylated, deacetylated, and diacetylated sophorolipids in acid...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Huang TT,Wages JM

    更新日期:2016-04-01 00:00:00

  • Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1.

    abstract::Growth conditions for mass production of luciferase-bacterial magnetic particles (BMPs) by a recombinant Magnetospirillum magneticum AMB-1 were investigated in a pH-regulated fed-batch culture system. Enrichment of growth medium with L-cysteine, yeast extract and polypeptone enhanced both bacterial growth and BMP prod...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Yang C,Takeyama H,Tanaka T,Matsunaga T

    更新日期:2001-07-05 00:00:00

  • CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS.

    abstract::Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae's strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a s...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Hou Y,Liu Z,Zhao Y,Chen S,Zheng Y,Chen F

    更新日期:2016-06-01 00:00:00

  • Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants.

    abstract::The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Lin L,Chien HR,Wang W,Hwang T,Fu H,Hsu W

    更新日期:2000-10-01 00:00:00

  • Enzymatic cascade biosynthesis reaction of musky macrolactones from fatty acids.

    abstract::Musky macrolactones are an important group of compounds used in high-valued perfumery. An enzymatic cascade reaction including cytochrome P450 hydroxylase and lipase was explored to biosynthesize musky macrolactones. Firstly, fatty acids were hydroxylated by P450 hydroxylase to produce the corresponding ω-hydroxy fatt...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Meng S,Guo J,Li Z,Nie K,Xu H,Tan T,Liu L

    更新日期:2019-12-01 00:00:00

  • Use of raw glycerol to produce oil rich in polyunsaturated fatty acids by a thraustochytrid.

    abstract::Glucose is the typical carbon source for producing microbial polyunsaturated fatty acids (PUFA) with single cell microorganisms such as thraustochytrids. We assessed the use of a fish oil derived glycerol by-product (raw glycerol), produced by a fish oil processing plant, as a carbon source to produce single cell oil ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Scott SD,Armenta RE,Berryman KT,Norman AW

    更新日期:2011-03-07 00:00:00

  • Enzyme reaction engineering: synthesis of antibiotics catalysed by stabilized penicillin G acylase in the presence of organic cosolvents.

    abstract::By using very active and very stable penicillin G acylase (PGA)--agarose derivatives we have studied the industrial design of equilibrium-controlled synthesis of lactamic antibiotics. In the presence of high concentrations of organic cosolvents we have carried out the direct enzymatic condensation of phenylacetic acid...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Fernandez-Lafuente R,Rosell CM,Guisán JM

    更新日期:1991-11-01 00:00:00

  • Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.

    abstract::Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those obser...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Pérez de los Santos AI,Cayetano-Cruz M,Gutiérrez-Antón M,Santiago-Hernández A,Plascencia-Espinosa M,Farrés A,Hidalgo-Lara ME

    更新日期:2016-02-01 00:00:00

  • Cellulase-lignin interactions-the role of carbohydrate-binding module and pH in non-productive binding.

    abstract::Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme-lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulas...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Rahikainen JL,Evans JD,Mikander S,Kalliola A,Puranen T,Tamminen T,Marjamaa K,Kruus K

    更新日期:2013-10-10 00:00:00