In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.
Biotechnology ; Enzymes ; chemistry ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism ; Synthetic Biology

Besides the catalytic domain, some xylanases contained a non-catalytic domain which is named as carbohydrate binding module (CBM). CBM can be used to improve their binding-ability to insoluble substrates. We illustrated the importance of CBM by reviewing the source of CBMs, type of families, features of binding to insoluble substrates, specific amino acids involved in substrate-binding, linker peptides connecting the catalytic domain, and the effect of CBMs on xylanase thermostability. CBM is important for xylanase to break down complicate carbohydrates. Perspectives on engineering xylanase activity according to the characteristics of CBMs were given.
Binding Sites ; Carbohydrate Metabolism ; Catalysis ; Endo-1,4-beta Xylanases ; metabolism ; Multienzyme Complexes ; chemistry ; Substrate Specificity

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.
Adenosine Triphosphate ; metabolism ; Arylamine N-Acetyltransferase ; metabolism ; Cardiolipins ; metabolism ; Electron Transport ; Humans ; Mitochondria ; enzymology ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism

OBJECTIVESTo investigate the relationships between proteasome active center LMP7 subunit and the occurrence and development of alcoholic liver disease.

METHODSEighty male Wistar rats, 170 to 190 g, were randomly divided into two groups: a model group (60 rats) and a control group (20 rats). The model group was given alcoholic intragastric administration plus an olive oil diet. Gavage, twice a day, was used to administer ethanol (30%) in a dose of 4 g/kg/d to the model group rats in the first 4 weeks. In the next 4 weeks, 40% ethanol in a dose of 5 g/kg/d was used, and then in the last 4 weeks, 50% ethanol in a dose of 6 g/kg/d was used. After infusion for 12 weeks, 15 rats (fatty liver group) were sacrificed. Others were divided into two groups; one was the hepatitis group with continued alcohol intragastric administration, the other was the hepatitis control group, receiving equal amounts of normal saline. Both groups were sacrificed after 4 weeks. By HE staining, histological pathology of the rat livers was analyzed. The expression of proteasome LMP7 subunit mRNA was examined by reverse transcription and real-time PCR. The content of LMP7 subunit protein was determined by Western blot.

RESULTSThe LMP7 mRNA level of the fatty liver group was 36% of the control group. The level of the hepatitis control group was 51% of the control group. The level of the hepatitis group was the lowest, which was only 26% of the control group. Western blot results showed that the level of the LMP7 protein content of the control group was 0.50+/-0.01; the level was 0.39+/-0.02 of the fatty liver group; 0.30+/-0.04 of the hepatitis group, and 0.38+/-0.02 of the hepatitis control group. The differences of the LMP7 protein content and mRNA expression correlated with the severity of the pathological alterations of the livers.

CONCLUSIONSThe proteasome LMP7 mRNA expression and protein content decreased in the alcoholic liver group. It may be one of the factors responsible for the decreased activity of proteasome and may play an important role in the pathogenesis of alcoholic liver disease.

Animals ; Liver ; pathology ; Liver Diseases, Alcoholic ; metabolism ; pathology ; Male ; Multienzyme Complexes ; metabolism ; Proteasome Endopeptidase Complex ; Rats ; Rats, Wistar

Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.
Acetyltransferases/metabolism ; Acetyltransferases/immunology ; Acetyltransferases/genetics* ; Animal ; Baculoviridae/genetics ; Cardiomyopathy, Congestive/immunology ; Cattle ; Human ; Immune Sera ; Insects/cytology* ; Ketone Oxidoreductases/metabolism ; Ketone Oxidoreductases/immunology* ; Ketone Oxidoreductases/genetics* ; Liver Cirrhosis, Biliary/immunology ; Multienzyme Complexes/metabolism ; Multienzyme Complexes/immunology* ; Multienzyme Complexes/genetics* ; Protein Engineering/methods ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/immunology ; Recombinant Proteins/genetics*

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.
Absorption ; Aflatoxin B1 ; metabolism ; toxicity ; Animals ; Chickens ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Multienzyme Complexes ; chemistry ; metabolism ; Rats ; Temperature ; Toxicity Tests

pHZ1080, an E. coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces. A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp. FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067. The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E. coli. The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot. The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E. coli and Streptomyces.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Multienzyme Complexes ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics ; Temperature

Adiponectin, derived mainly from white adipose tissue, regulates glucose and fatty acid metabolism and has anti-inflammatory and anti-atherosclerotic properties. The decrease in plasma adiponectin concentration contributes to the development of metabolic and cardiovascular diseases. AMP-activated protein kinase (AMPK) is a serine/threonine kinase which plays an important role in regulating many cellular processes, particularly pathways involved in cellular energy status. AMPK is now recognized as a fuel gauge in mammalian cells. Adiponectin activates AMPK phosphorylation and then promotes ATP-generating pathways in heart, including glucose transport, glycolysis, and fatty acid oxidation. The recent evidence has shown that AMPK activation has an important role in the vasculature where it may exert anti-atherosclerotic effects. Phosphorylation of AMPK induced by adiponectin inhibits protein synthesis, and may be an adaptive response to pathological cardiac hypertrophy. AMPK also has a cardioprotective role against myocardial injury and apoptosis in the ischemic heart. This review will discuss the role of AMPK in adiponectin-mediated protective properties of cardiovascular diseases.
AMP-Activated Protein Kinases ; metabolism ; Adiponectin ; physiology ; Animals ; Cardiomegaly ; Cardiovascular Diseases ; physiopathology ; Energy Metabolism ; Glucose ; Heart ; Humans ; Multienzyme Complexes ; Phosphorylation ; Protein-Serine-Threonine Kinases

Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm.
Aldose-Ketose Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Mevalonic Acid ; metabolism ; Multienzyme Complexes ; genetics ; Oxidoreductases ; genetics ; Phylogeny ; RNA ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Taxus ; genetics

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Chromosomes, Bacterial ; Erythromycin ; analogs & derivatives ; biosynthesis ; chemistry ; Genetic Engineering ; Ketolides ; Molecular Structure ; Multienzyme Complexes ; genetics ; Saccharopolyspora ; enzymology ; genetics ; metabolism