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












  • Kinetic resolution of sec-alcohols catalysed by Candida antarctica lipase B displaying Pichia pastoris whole-cell biocatalyst.

    abstract::Kinetic resolution of sec-alcohols is a green process with biocatalyst. Candida antarctica lipase B (CALB) displayed on Pichia pastoris cell-surface (Pp-CALB) was characterized in kinetic resolution of sec-alcohols with different structures. The reaction parameters including acyl donors, molar ratio of substrates, sol...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Zhang K,Pan Z,Diao Z,Liang S,Han S,Zheng S,Lin Y

    更新日期:2018-03-01 00:00:00

  • Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B.

    abstract::This study examines a novel method to reduce the probability of disulfide mismatches during the refolding process by the replacement of cysteines within a protein. Specifically, Cys383 of recombinant rat procarboxypeptidase B was replaced by other amino acids to increase the refolding efficiency in vitro. Mutants C383...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Li S,Zhang L,Wu Q,Xin A,Zhao J,Fan L

    更新日期:2011-07-10 00:00:00

  • Effect of immobilization on the activity of rat hepatic microsomal cytochrome P450 enzymes.

    abstract::Cytochrome P450 enzyme preparations were obtained from phenobarbital-treated rats. On immobilization in Romicon PM 10 polysulfone anisotropic hollow fibers, 20-25% of the original activity remained. The immobilization procedure did not significantly alter the Km of the preparations when 7-ethoxycoumarin was the substr...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Fernandez-Salguero P,Gutierrez-Merino C,Bunch AW

    更新日期:1993-02-01 00:00:00

  • The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

    abstract::To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzy...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Cintra LC,da Costa IC,de Oliveira ICM,Fernandes AG,Faria SP,Jesuíno RSA,Ravanal MC,Eyzaguirre J,Ramos LP,de Faria FP,Ulhoa CJ

    更新日期:2020-02-01 00:00:00

  • UV-Vis detection of hydrogen peroxide using horseradish peroxidase/copper phosphate hybrid nanoflowers.

    abstract::A colorimetric platform based on enzyme-inorganic hybrid nanoflowers was used for ultraviolet-visible detection of hydrogen peroxide (H2O2). The hybrid nanoflowers were prepared via incubation at room temperature using horseradish peroxidase (HRP) and copper phosphate (Cu3(PO4)2) as the organic and inorganic component...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Yang C,Zhang M,Wang W,Wang Y,Tang J

    更新日期:2020-10-01 00:00:00

  • Cellulose oxidation and bleaching processes based on recombinant Myriococcum thermophilum cellobiose dehydrogenase.

    abstract::Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) was expressed in Pichia pastoris using the pPICZαA expression vector under the control of methanol inducible AOX promoter. The purified recombinant MtCDH with a specific activity of 3.1 Umg(-1) was characterized to obtain kinetic constants for various carbohydr...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Flitsch A,Prasetyo EN,Sygmund C,Ludwig R,Nyanhongo GS,Guebitz GM

    更新日期:2013-01-10 00:00:00

  • Structural and functional consequences of replacement of His403 with Arg near the catalytic site of Anoxybacillus flavithermus cyclomaltodextrinase.

    abstract::The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, β- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue prot...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Mirzaee Z,Jafarian V,Shirdel SA,Khalifeh K

    更新日期:2019-12-01 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

  • The influence of hexoses addition on the fermentation of d-xylose in Debaryomyces hansenii under continuous cultivation.

    abstract::The effect of hexoses (glucose and galactose) addition to the feed xylose mineral medium of Debaryomyces hansenii chemostat cultures grown at a constant dilution rate of 0.055 h(-1) was studied. Xylitol was the major product detected amongst all tested conditions. The maximal values for xylitol yield and volumetric pr...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Tavares JM,Duarte LC,Amaral-Collaço MT,Gírio FM

    更新日期:2000-06-01 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

  • Flow cytometric immunoassay using E. coli with autodisplayed Z-domains.

    abstract::Recently, we reported a highly sensitive immunoassay using Escherichia coli cells with autodisplayed Z-domains. In this work, E. coli cells with autodisplayed Z-domains were applied to the flow-cytometry-based simultaneous detection of multiple analytes. The E. coli cells were doubly transfected to express a fluoresce...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Park M,Bong JH,Yoo G,Jose J,Kang MJ,Pyun JC

    更新日期:2013-08-15 00:00:00

  • Enhancement of neuroprotection, antioxidant capacity, and water-solubility of crocins by transglucosylation using dextransucrase under high hydrostatic pressure.

    abstract::Crocin, one of the major carotenoid pigments of Crocus sativus (saffron), is responsible for antioxidant activity, neuroprotection, and the inhibition of tumor cell proliferation. In order to improve the functionality of crocin, α-glucosyl-(1→6)-trans-crocins (C-Gs) were synthesized using sucrose and dextransucrase fr...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Mok IK,Nguyen TTH,Kim DH,Lee JW,Lim S,Jung HY,Lim T,Pal K,Kim D

    更新日期:2020-10-01 00:00:00

  • Kinetics of sequential nitrification and denitrification processes.

    abstract::Kinetics of nitrification and denitrification of synthetic wastewater was investigated by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter for denitrification. Glucose solution was fed to the denitrification column to supply carbon source. Reactors were...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Dinçer AR,Kargi F

    更新日期:2000-07-01 00:00:00

  • Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis.

    abstract::Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. t...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Jung IP,Cho JH,Koo BS,Yoon MY

    更新日期:2015-10-01 00:00:00

  • The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase.

    abstract::Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). Th...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Sanhueza C,Carvajal G,Soto-Aguilar J,Lienqueo ME,Salazar O

    更新日期:2018-06-01 00:00:00

  • Glucosylation of the flavonoid, astragalin by Leuconostoc mesenteroides B-512FMCM dextransucrase acceptor reactions and characterization of the products.

    abstract::Astragalin (kaempferol-3-O-β-D-glucopyranoside, Ast) glucosides were synthesized by the acceptor reaction of a dextransucrase produced by Leuconostoc mesenteroides B-512FMCM with astragalin and sucrose. Each glucoside was purified and their structures were assigned as kaempferol-3-O-β-D-glucopyranosyl-(1→3)-O-α-D-gluc...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Kim GE,Kang HK,Seo ES,Jung SH,Park JS,Kim DH,Kim DW,Ahn SA,Sunwoo C,Kim D

    更新日期:2012-01-05 00:00:00

  • Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

    abstract::The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condit...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Chen H,Shao M,Li H

    更新日期:2014-03-05 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

  • Growth of 'Candidatus Liberibacter asiaticus' in a host-free microbial culture is associated with microbial community composition.

    abstract::'Candidatus Liberibacter asiaticus' ('Ca. L. asiaticus'), the suspected causative agent of citrus greening disease, is one of many phloem-restricted plant pathogens that have not been isolated and grown in an axenic culture. In this study, infected Asian citrus psyllids were used to prepare a host-free source of 'Ca. ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Molki B,Call DR,Ha PT,Omsland A,Gang DR,Lindemann SR,Killiny N,Beyenal H

    更新日期:2020-12-01 00:00:00

  • Biosensors for process control.

    abstract::Biosensors have been extensively studied during the last 20 years, and a myriad of laboratory biosensors have been developed. Improvements are required in biosensor design and performance before they become widely accepted in industrial process monitoring. However, as the biotechnology industry expands, biosensors may...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章,评审


    authors: Brooks SL,Higgins IJ,Newman JD,Turner AP

    更新日期:1991-12-01 00:00:00

  • Direct and selective enzymatic synthesis of trehalose unsaturated fatty acid diesters and evaluation of foaming and emulsifying properties.

    abstract::Trehalose diesters are Gemini-type surfactants that might have better surface activity than conventional surfactants. A one-step method for the preparation of trehalose unsaturated fatty acid diesters has been successfully developed. The yield of trehalose diester of different unsaturated fatty acids was between 78 % ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Ji S,Jia C,Cao D,Li S,Zhang X

    更新日期:2020-05-01 00:00:00

  • Factors relevant to the production of (R)-(+)-glycidol (2,3-epoxy-1-propanol) from racemic glycidol by enantioselective oxidation with Acetobacter pasteurianus ATCC 12874.

    abstract::Acetobacter pasteurianus oxidizes glycidol with high activity, comparable to the oxidation of ethanol. The organism has a preference for the S-enantiomer, and the kinetic resolution process obeys a simple relationship, indicating an enantiomeric ratio (E) of 19. The compound is converted into glycidic acid, although a...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Geerlof A,Jongejan JA,van Dooren TJ,Racemakers-Franken PC,van den Tweel WJ,Duine JA

    更新日期:1994-12-01 00:00:00

  • Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest.

    abstract::Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and qua...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章,评审


    authors: Cologna NMD,Gómez-Mendoza DP,Zanoelo FF,Giannesi GC,Guimarães NCA,Moreira LRS,Filho EXF,Ricart CAO

    更新日期:2018-02-01 00:00:00

  • Effect of Pluronic F-68 on the mechanical properties of mammalian cells.

    abstract::The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility m...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Zhang Z,al-Rubeai M,Thomas CR

    更新日期:1992-12-01 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

  • Acyl transfer reactions catalyzed by native and modified alpha-chymotrypsin in acetonitrile with low water content.

    abstract::The characterization of the S' subsite specificity of native and ethylated alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl donor, we investigated the partitioning of acyl-chymotrypsins between water and amino acid and peptide-derived ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Cerovský V,Jakubke HD

    更新日期:1994-07-01 00:00:00

  • ZnS quantum dots as pH probes for study of enzyme reaction kinetics.

    abstract::Water soluble ZnS quantum dots (QDs) modified by mercaptoacetic acid (MAA) were used to determinate proton concentration in aqueous solutions by fluorescence spectroscopic technique. The results showed that the fluorescence of the water-soluble QDs could be quenched by proton concentration and the fluorescence intensi...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Wu D,Chen Z

    更新日期:2012-06-10 00:00:00

  • Catalytic synthesis of 2,5-bis(hydroxymethyl)furan from 5-hydroxymethylfurfual by recombinant Saccharomyces cerevisiae.

    abstract::2,5-Bis(hydroxymethyl)furan (BHMF) is a versatile building block in the synthesis of polymers, fuels, and macrocycle polyethers. In this work, alcohol dehydrogenases (ADHs) were identified from Meyerozyma guilliermondii SC1103 and were heterologously expressed in Saccharomyces cerevisiae for the synthesis of BHMF from...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Xia ZH,Zong MH,Li N

    更新日期:2020-03-01 00:00:00

  • Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli.

    abstract::The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful ...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Grant C,Woodley JM,Baganz F

    更新日期:2011-05-06 00:00:00

  • A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms.

    abstract::l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and b...

    journal_title:Enzyme and microbial technology

    pub_type: 杂志文章


    authors: Mihooliya KN,Nandal J,Swami L,Verma H,Chopra L,Sahoo DK

    更新日期:2017-12-01 00:00:00