Encapsulation of Trigonopsis variabilis D-amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme

A one-step procedure of immobilizing soluble and aggregated preparations of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is reported where carrier-free enzyme was entrapped in semipermeable microcapsules produced from the polycation poly(methylene-co-guanidine) in combination with CaCl2 and the polyanions alginate and cellulose sulfate. The yield of immobilization, expressed as the fraction of original activity present in microcapsules, was approximately 52 +/- 5%. The effectiveness of the entrapped oxidase for O2-dependent conversion of D-methionine at 25 degrees C was 85 +/- 10% of the free enzyme preparation. Because continuous spectrophotometric assays are generally not well compatible with insoluble enzymes, we employed a dynamic method for the rapid in situ estimation of activity and relatedly, stability of free and encapsulated oxidases using on-line measurements of the concentration of dissolved O2. Integral and differential modes of data acquisition were utilized to examine cases of fast and slow inactivation of the enzyme, respectively. With a half-life of 60 h, encapsulated TvDAO was approximately 720-fold more stable than the free enzyme under conditions of bubble aeration at 25 degrees C. The soluble oxidase was stabilized by added FAD only at temperatures of 35 degrees C or greater.     

透明颤菌血红蛋白对两种D-氨基酸氧化酶的不同影响及机理研究

D-氨基酸氧化酶是两步酶法制备7-氨基头孢烷酸(7-ACA)这一半合成头孢类抗生素的主要前体的关键酶.它催化的反应是需氧反应,反应体系的溶氧水平是酶活的限制因素之一.我们发现将纯化的透明颤菌血红蛋白(VHb)分别添加到三角酵母来源(TvDAO)和红酵母来源(RgDAO)的D-氨基酸氧化酶的纯酶中,可提高这两种氧化酶的活力35%和48%.细菌双杂交实验证明,透明颤菌血红蛋白与RgDAO有相互作用,而与TvDAO没有关联.这说明透明颤菌血红蛋白对氧化酶活力的促进是由于自身向氧化酶提供游离氧,而且它与氧化酶之间的相互作用可以增强这种效果.我们可以利用透明颤菌血红蛋白的这种性质把它作为氧化酶酶促反应的添加剂,提高酶促反应的效率,如果该氧化酶与之有相互作用,效果会更加显著.     

Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.     

Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates

Insoluble protein particles showing high specific enzyme activity are potentially useful biocatalysts. The commercialized crosslinked enzyme crystals and aggregates have the disadvantage that their preparation requires isolation of the protein before the critical precipitation step. We introduce a novel concept of controlled precipitation in vivo in which the target enzyme is fused to the cellulose-binding domain (CBD) of Clostridium cellulovorans, and expression in Escherichia coli is performed under conditions that induce selective pull down of the folded chimeric protein via intermolecular self-aggregation of the CBD. The case of D-amino acid oxidase from Trigonopsis variabilis shows that upon fusion of the CBD to its N-terminus, the otherwise mainly soluble recombinant enzyme was quantitatively precipitated in protein particles, which displayed 40% of the specific activity of the highly purified oxidase. By contrast, inclusion bodies derived from an enzyme chimera, which harbored a C-terminal peptide tag, showed only little oxidase activity (相似文献   

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

Electrostatic immobilization of pectinase on negatively charged AOT-Fe3O4 nanoparticles

Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotechnology during the past decade. The present study suggests MNPs negatively charged by docusate sodium salt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant which can stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppositely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration, aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization. Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme loading of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentration is significantly important on both loading and activity of immobilized enzyme, while pH and AOT concentration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recovered easily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immobilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated with immobilization by electrostatic adsorption, the medium pH was changed to improve the release of protein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis.     

Ion exchange immobilization of changed beta-galactosidase fusions for lactose hydrolysis

The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. (c) 1994 John Wiley & Sons, Inc.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant K_m values of EP-PEI-CCA and EP-EDA-CCA were 22?mM and 30?mM, respectively, which were lower than that of the free enzyme (43?mM), suggesting that the support’s zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Functionalization of catalase for a photochemical immobilization on poly(ethylene terephthalate)

The enzyme catalase (EC 1.11.1.6) was covalently immobilized on textile carrier fabrics made of poly(ethylene terephthalate) (PET) by a novel combined wet chemical and photochemical process. The functionalization of catalase with allyl groups succeeds in a wet chemical treatment of the enzyme with allylglycidylether. This modified enzyme was bonded covalently to the textile material by a photochemical immobilization using a monochromatic excimer UV lamp (222 nm). Using this two-step procedure nearly 60 mg enzyme/g carrier could be fixed durably. The efficiency of the immobilization products was investigated by measuring the enzymatic decomposition of hydrogen peroxide in comparison to the free enzyme. The relative activity of the catalase after the immobilization was nearly 5% compared to the free, not fixed enzyme; however, even after 30 reuses, the modified and immobilized catalase still showed a distinct activity.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.