Formation of 6-O-alpha-maltosylcyclomalto-oligosaccharides by transfer action of three debranching enzymes

O-Maltosylcyclomaltohexaoses (G2-cG6) were formed in yields of 24.3 and 23.2 mmol from 40 mmol of alpha-maltosyl fluoride (alpha-G2F) and 90 mmol of cyclomaltohexaose (cG6) by the transfer action of pullulanase from Aerobacter aerogenes (A-pullulanase) and isoamylase from Pseudomonas amyloderamosa, respectively. These yields were three times that given by pullulanase from Bacillus acidopullulyticus (B-pullulanase). The yields of O-maltosylcyclomalto-oligosaccharides were changed according to the origin of the enzymes and the kind of cyclomalto-oligosaccharide (cG6, cG7, or cG8) used as the acceptor. By the reaction with 40 mmol of alpha-G2F and 90 mmol of cG6, 20 mmol of alpha-G2F and 30 mmol of cG7, or 40 mmol of alpha-G2F and 90 mmol of cG8, the amounts of O-maltosylcyclomalto-oligosaccharides produced and the transfer ratios of alpha-G2F to the acceptors were as follows. By A-pullulanase, 24.3 mmol of G2-cG6 was produced in a 60.8% transfer ratio, whereas the yields of G2-cG7 and G2-cG8 were 1.7 mmol (8.5%) and 8.4 mmol (21.0%), respectively. The yields of G2-cG6, G2-cG7, and G2-cG8 by B-pullulanase were 8.8 mmol (22.0%), 1.2 mmol (6.0%), and 11.7 mmol (29.3%), respectively. In the case of isoamylase, G2-cG7 (9.2 mmol, 46.0%) and G2-cG8 (20.9 mmol, 52.3%) were produced, as much as for G2-cG6 (23.2 mmol, 58.0%). It was suggested that the difference in the amounts of G2-cG6 produced by these three debranching enzymes is based on the difference in the mode of action on the alpha-G2F used as the substrate, either a transfer action or a hydrolytic action.     

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl- Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,and Isolation and Characterization of the Products

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)₂-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)₂-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(6³-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)₂-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)₂-βCDs were identified as 6¹,6²-, 6¹,6³- and 6¹,6⁴-di-O-(6³-O-α-D-mannosyl-maltotriosyl)-βCD.     

Synthesis of Maltosyl(α1→6)cyclodextrins through the Reverse Reaction of Thermostable Bacillus acidopullulyticus Pullulanase

Maltosyl(α1→6)α-, β or γ-cyclodextrin was synthesized from maltose and α-, β- or γ- cyclodextrin, respectively, using Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). More than 40% of each cyclodextrin substrate was converted to the corresponding maltosyl(α1→6)cyclodextrin under the conditions given below; the combined concentration of maltose and cyclodextrin was 70 ~ 75 % (w/w), the molar ratio of maltose to cyclodextrin was 9~18, and the amount of pullulanase was 100~200units/g of cyclodextrin. The optimum pH and temperature for the formation of maltosyl(α1→6)cyclodextrins were 4.0—4.5 and 60~70°C, respectively. Each maltosyl(α1→6)-cyclodextrin produced was separated from noncyclic saccharides, maltose and branched tetraose, by methanol and ethanol precipitations. The maltosyl(α1→6)cyclodextrins were further purified by gel filtration on a Toyopearl HW 40 S column and crystallization from aqueous (for maltosyl(α1→6)β-cyclodextrin) or methanol (for maltosyl(α1→6)β-cyclodextrin) solution. From 10 g each of the corresponding cyclodextrin, the yields of the purified maltosyl(α1→6)α-, β- and γ-cylcodextrins were 3.0 ~ 3.6 g, 2.5 ~ 2.8g and 2.2 ~ 2.5 g, respectively. Identification of the maltosyl(α1-6)cyclo-dextrins was performed by means of hydrolysis with Klebsiella pneumoniae pullulanase, methyla- tion analysis and ¹³C-NMR analysis.     

An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities

A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k _cat/K _m , 342.34 s^?1?mL?mg^?1) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH?5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl₂. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-O-maltooligosyl-β-CD were [6)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1→]_n, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase.     

Encapsulation of Citral Isomers in Extracted Lemongrass Oil with Cyclodextrins: Molecular Modeling and Physicochemical Characterizations

The complexation between two isomers of citral in lemongrass oil and varying types of cyclodextrins (CDs), α-CD, β-CD, and HP-β-CD, were studied by molecular modeling and physicochemical characterization. The results obtained revealed that the most favorable complex formation governing between citrals in lemongrass oil and CDs were found at a 1:2 mole ratio for all CDs. Complex formation between E-citral and CD was more favorable than between Z-citral and CD. The thermal stability of the inclusion complex was observed compared to the citral in the lemongrass oil. The release time course of citral from the inclusion complex was the diffusion control, and it correlated well with Avrami’s equation. The release rate constants of the E- and Z-citral inclusion complexes at 50 °C, 50% RH were observed at 1.32×10^?2 h^?1 and 1.43×10^?2 h^?1 respectively.     

Physicochemical and biological properties of 2-O-α-d-galactosyl-cyclomaltohexaose (α-cyclodexterin) and -cyclomaltoheptaose (β-cyclodextrin)

The physicochemical and biological properties of the new branched cyclomaltooligosaccharides (cyclodextrins; CDs), 2-O-α-d-galactosyl-cyclomaltohexaose (2-O-α-d-galactosyl-α-cyclodextrin, 2-Gal-αCD) and 2-O-α-d-galactosyl-cyclomaltoheptaose (2-O-α-d-galactosyl-β-cyclodextrin, 2-Gal-βCD), were investigated. The formation of inclusion complexes of 2-Gal-CDs with various kinds of guest compounds (clofibrate, cholesterol, cholecalciferol, digitoxin, digitoxigenin, and prostaglandin A₁) was examined by a solubility method, and the results were compared with those of non-branched CDs and other 6-O-glycosyl-CDs such as 6-O-α-d-galactosyl-CDs, 6-O-α-d-glucosyl-CDs, and 6-O-α-maltosyl-CDs. The inclusion abilities of 2-Gal-αCD for clofibrate and prostaglandin A₁, and 2-Gal-βCD for clofibrate, cholecalciferol, cholesterol, and digitoxigenin were markedly weaker than those of non-branched CD and other 6-O-glycosyl-CDs in each series, probably because of a steric hindrance caused by the α-(1→2)-galactoside linkage. The hemolytic activities of 2-Gal-CDs on human erythrocytes were the lowest among each CD series, and the compounds showed negligible cytotoxicity towards Caco-2 cells up to at least 100 mM.     

Effects of Moranoline, 4-O-α-D-Glucopyranosylmoranoline and Their N-Substituted Derivatives on Thermostability of Cyclodextrin Glycosyltransferase,Glucoamylase, and β-Amylase

In repeated glycosylmoranolines synthetic reaction at 55°C, cyclodextrin glycosyltransferase (CGT-ase, EC 2.4.1.19) from Bacillus stearothermophilus retained its activity for more than 600 days. A main stabilizing compound. was found to be 4-O-α-D-glucopyranosylmoranoline.

The thermostabilizing activities of moranoline, 4-O-α-D-glucopyranosylmoranoline, and their N-substituted derivatives were studied. Moranoline and its N-substituted derivatives stabilized glucoamylase. 4-O-α-D-Glucopyranosylmoranoline and its N-substituted derivatives stabilized CGT-ase and β-amylase.     

Modulation of Ca Activity in Cardiomyocytes through Caveolae-Gαq Interactions

Cardiomyocytes have a complex Ca²⁺ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca²⁺ activity can be regulated by the phospholipase Cβ–Gα_q pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα_q, which could affect Ca²⁺ signals. Here, using fluorescence imaging and correlation techniques we show that Gα_q-Gβγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gβγ subunits from caveolae with a concurrent stabilization of activated Gα_q by caveolin-3 (Cav3). These cells show oscillating Ca²⁺ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα_q association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα_q is responsible for this Ca²⁺ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca²⁺ activity in cardiomyocytes.     

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular α‐(1,4)‐linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 α‐D‐glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD₁₂) as the template molecule. The imprinted CGTase was stabilized by cross‐linking of the derivatized protein. A high proportion of CD₁₂ and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD₁₂‐forming cyclization reaction, while decreased k_cat/K_m values of the reverse ring‐opening reaction were observed. The maximum yield of CD₁₂ was obtained when the imprinted CGTase was reacted with amylose at 40°C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG₆ < cG₇ < cG₈). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-α-maltotriosylmaltotriose (branched G₆) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G₆ was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and ¹³C-n.m.r. spectroscopic analyses.     

Kinetics of racemization of (+)- and (−)-diethylpropion: Studies in aqueous solution,with and without the addition of cyclodextrins,in organic solvents and in human plasma

The configurational stability of (+)- and (−)-diethylpropion [(+)- and (−)-2-(diethyl)-1-phenyl-1-propanone or (+)- and (−)-DEP ] was investigated systematically from chemical, pharmaceutical, and pharmacological aspects. The enantiomeric ratio was monitored directly with a recently developed stability-indicating enantioselective HPLC method. In aqueous solutions, the rate of racemization increased non-linearly with increasing pH and with increasing phosphate buffer concentration. The racemization rate showed a positive slope with increasing temperature and decreasing ionic strength. The racemization rates of (+)- and (−)-DEP in the presence of cyclodextrins (CDs) did not differ significantly. CDs that were added to (+)- and (−)-DEP in a molar ratio 5:1 showed the following effects after dissolution in 10 mM phosphate buffer (final pH 6.7): sulfobutyl ether-β-CD (SBE-β-CD) and methylated-β-CD (Me-β-CD) retarded racemization; whereas hydroxypropyl-β-CD (HP-β-CD), acetyl-γ-CD (Ac-γ-CD), acetyl-β-CD (Ac-β-CD), γ-CD, and β-CD showed a weak destabilising effect. In contrast to the described CDs, α-CD distinctly accelerated the rate of racemization. The configurational stability of (+)- and (−)-DEP was also studied under physiological conditions. The half-life of racemization in heparinised human plasma was for both enantiomers determined to be approximately 23–25 min. In phosphate buffer (10 mM, pH 7.4), rac-DEP showed a high, but unselective affinity towards human α₁-acid glycoprotein (orosomucoid) immobilised on silica (Chiral AGP). The rate of racemization of the free base of (−)-DEP dissolved in organic solutions generally increases with the polarity of the solvating agent. Chirality 10:307–315, 1998. © 1998 Wiley-Liss, Inc.     

Enzymatic synthesis of dimaltosyl-beta-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme

Di-O-α-maltosyl-β-cyclodextrin ((G2)₂-β-CD) was synthesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-β-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-β-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-β-CD, suggesting that TreX transferred the maltosyl residue of a G2-β-CD to another molecule of G2-β-CD by forming an α-1,6-glucosidic linkage. When [¹⁴C]-maltose and maltosyl-β-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-β-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6¹,6³- and 6¹,6⁴-dimaltosyl-β-CD. Inhibition studies with β-CD on the transglycosylation activity revealed that β-CD was a mixed-type inhibitor, with a K_i value of 55.6 μmol/mL. Thus, dimaltosyl-β-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of β-CD. Our findings suggest that the high yield of (G2)₂-β-CD from G2-β-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of β-CD.     

Crystal and molecular structures of cyclomaltoheptaose inclusion complexes with HI and with methanol

β-Cyclodextrin (β-CD; cyclomaltoheptaose; cyclohepta-amylose; C₄₂H₇₀O₃₅) crystallises from aqueous solutions of HI and of MeOH in the form of stout prisms, which are isomorphous to each other with monoclinic space-group P2₁; cell constants for C₄₂H₇₀O₃₅ · 2HI · 8 H₂O: a = 21.25(3), b = 10.28(2), c = 15.30(2) Å, β = 113.25(9)°, and Z = 2; and for C₄₂H₇₀O₃₅ · MeOH · 6.5 H₂O: a = 21.03(3), b = 10.11(2), c = 15.33(2) Å, β = 111.02(9)°, and Z = 2. X-ray counter data were used to determine the structures of both crystals, which belong to the cage type, with β-CD molecules in nearly identical, “round” shapes. In the HI complex, one I^- is located inside, and one outside, the β-CD cavity; in the MeOH complex, the MeOH is within the cavity. The cavity is closed at the O-2,O-3 side by adjacent β-CD molecules, and at the O-6 side by water molecules hydrogen-bonded to the guest and to surrounding β-CD molecules. Interstices between β-CD molecules are filled by water of hydration molecules in distorted co-ordination.     

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G₁), maltose (G₂), maltotriose (G₃), maltoheptaose (G₇), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (μ_max) were observed with glucose, γ-CD and soluble starch (μ_max values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×10¹¹ CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G₇ (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G₂ and G₃ saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.     

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (a_w) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17–42°C and 0.90–0.99 a_w. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 a_w. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B₁ or G₁ biosynthesis. The temperature optima of aflatoxin B₁ and G₁ were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G₁, 20–30°C) or above (aflatoxin B₁, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B₁ (aflR) or G₁ (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G₁ biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B₁ biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B₁, whereas it was water activity for G₁ biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.     

Antiseptic cyclodextrin-functionalized hydrogels and gauzes for loading and delivery of benzalkonium chloride

Prevention and management of wound infections receive a lot of attention, since the presence of micro-organisms interferes with the wound-healing process. The aim of this work was to use cyclodextrins (CDs) to endow hydrogels and gauzes with the ability to take up antiseptics and sustain their delivery for several hours. Benzalkonium chloride (BzCl) can form inclusion complexes with cross-linked CDs that regulate the release through an affinity-driven mechanism. Grafting of CDs to cotton gauzes using citric acid as the linker, at 190?°C and for 15?min, led to grafting yields of about 148%, much larger than those obtained at 180?°C or with shorter reaction times. Microbiological tests revealed that the BzCl-loaded networks can inhibit the growth of Staphylococcus epidermidis and Escherichia coli both on agar plates and in liquid medium. Furthermore, the antiseptic-loaded gauzes were able to inhibit biofilm formation by Staphylococcus aureus RN1HG pMV158GFP when applied in early stages of biofilm formation and could reduce the number of living cells in preformed biofilms grown in a chronic wound biofilm model. These findings highlight the role of CDs as main components of hydrogels and gauzes for the efficient delivery of antiseptics.     

Enantioselective host-guest complexation of Ru(II) trisdiimine complexes using neutral and anionic derivatized cyclodextrins

Enantioselective host-guest complexation between five racemic Ru(II) trisdiimine complexes and eight derivatized cyclodextrins (CDs) has been examined by NMR techniques. The appearance of non-equivalent complexation-induced shifts of between the Δ and Λ-enantionomers of the Ru(II) trisdiimine complexes and derivatized CDs is readily observed by NMR. In particular, sulfobutyl ether-β-cyclodextrin sodium salt (SBE-β-CD), R-naphtylethyl carbamate β-cyclodextrin (RN-β-CD), and S-naphtylethyl carbamate β-cyclodextrin (SN-β-CD) showed good enantiodiscrimination for all five Ru complexes examined, which indicates that aromatic and anionic derivatizing groups are beneficial for chiral recognition. The complexation stoichiometry between SBE-β-CD and [Ru(phen)₃]²⁺ was found to be 1:1 and binding constants reveal that Λ-[Ru(phen)₃]²⁺ binds more strongly to SBE-β-CD than the Δ-enantiomer. Correlations between this NMR method and separative techniques based on CDs as chiral discriminating agents (i.e., selectors) are discussed in detail.     

Action of Pseudomonas isoamylase on various branched oligo- and poly-saccharides

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 → 6)-α-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase. Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3)Pr. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz