Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

The molecular structures of some glucans from the cell walls of Schizosaccharomyces pombe

Extraction of the cell walls of Schizosaccharomyces pombe with dilute alkali at 4° yields a mixture of polysaccharides including galactomannan, (1→3)-α-d-glucan, and a branched (1→3)-β-d-glucan. The alkali-insoluble residue contains a lightly branched (1→3)-β-d-glucan, together with smaller amounts of an extremely highly branched (1→6)-β-d-glucan. The properties of the three distinct β-d-glucans are compared with those isolated from other yeasts.     

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α]_D²⁰ values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-d-Gal-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-[β-d-Gal-(1→6)]-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, and β-d-Gal-(1→3)-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.     

THE COMPLEX POLYSACCHARIDES OF THE RAPHID DIATOM PINNULARIA VIRIDIS (BACILLARIOPHYCEAE)1

A combination of carbohydrate analysis and atomic force microscopy (AFM) was used to characterize the polysaccharides of the pennate diatom, Pinnularia viridis (Nitzsch) Ehrenberg. Polymeric substances were fractionated into those in the spent culture medium (SCM) and those sequentially extracted from the cells with water at 45° C (WW), NaHCO₃ containing EDTA at 95° C (HB), and 1 M NaOH containing NaBH₄ at 95° C. Carbohydrate, protein, and sulfate were detected in all the fractions, but their relative proportions differed significantly. Nineteen sugars were identified, including pentoses, hexoses, 6‐deoxyhexoses, O‐methylated sugars, aminohexoses, and traces of uronic acids. To some extent, the same constituent monosaccharides and a proportion of the linkage patterns occurred in all four fractions, indicating the fractions contained a spectrum of highly heterogeneous but structurally related polysaccharides. Several carbohydrates were enriched in specific fractions. A soluble, partially substituted, 3‐linked galactan was slightly enriched in the SCM. The WW fraction was highly enriched in 3‐linked glucan, presumably derived from chrysolaminaran. Chemical and AFM data for the WW and HB fractions indicated that compositional differences were associated with substantial changes in the morphology and properties of the cell surface mucilage. Soluble polymers relatively enriched in fucose conferred a degree of softness and compressibility to the mucilage, whereas most of the mucilage comprised firmer more gelatinous polymers comparatively enriched in rhamnose. The frustule residue dissolved during extraction with NaOH, and a partially substituted 3‐linked mannan, together with relatively large amounts of protein, was obtained.     

Catalytic properties of endo-1,3-β-D-glucanase from the Vietnamese edible mussel Perna viridis

A β-1,3-glucanase with a molecular mass of 33 kDa was isolated in the homogeneous state from a crystalline stalk of the commercially available Vietnamese edible mussel Perna viridis. It hydrolyzes β-1,3-bonds in glucans and is capable of catalyzing the transglycosylation reaction. The β-1,3-glucanase has a K _m value of 0.3 mg/ml for the hydrolysis of laminaran and shows a maximum activity in the pH range from 4 to 6.5 and at 45°C. Its half-inactivation time is 180 min at 45°C and 20 min at 50°C. The enzyme was ascribed to glucan-endo-(1 → 3)-β-D-glucosidases (EC 3.2.1.39). The enzyme could be used in the structure determination of β-1,3-glucans and enzymatic synthesis of new carbohydrate-containing compounds.     

Glycan structures of ganoderans b and c,hypoglycemic glycans of ganoderma lucidum fruit bodies

Two hypoglycemic principles, ganoderans B and C, isolated from the fruit bodies of Ganoderma lucidum were shown to be peptidoglycans with M,s of 7400 and 5800, respectively. Physico-chemical and chemical studies demonstrated that the backbone and side chains of ganoderan B contain D-glucopyranosyl β-1 → 3 and β-1 → 6 linkages while those of ganoderan C contain D-glucopyranosyl β-1 → 3 and β-1 → 6 linkages and a D-galactopyranosyl α-1 → 6 linkage.     

Fractionation and characterization of chlorophyll and lignin from de-juiced Italian ryegrass (Lolium multifolrum) and timothy grass (Phleum pratense)

In this study, de-juiced Italian ryegrass and timothy grass were successfully fractionated into one chlorophyll rich fraction, two lignin fractions, two hemicellulosic preparations, and one cellulosic fraction by sequential processes with 80% ethanol containing 0.2% NaOH, 2.5% H₂O₂–0.2% EDTA containing 1.5% NaOH, and 2.5% H₂O₂–0.2% TAED containing 1.0% NaOH at 75 °C for 3 h, respectively. The yields of chlorophyll rich fraction and total lignin fraction were found to be 6.1 and 11.0% from de-juiced Italian ryegrass, and 12.7 and 13.2% from de-juiced timothy grass. It should be noted that the two chlorophyll rich fractions contained noticeable amounts of contaminated hemicelluloses (17.1–18.8%) and lignin, whereas the four lignin fractions contained only small amounts of bound hemicelluloses (5.6–8.6%). Nitrobenzene oxidation and NMR spectra of the lignin fraction revealed that the presence of both guaiacyl and syringyl units, as well as p-hydroxyphenyl structures. Small amounts of esterified p-coumaric acids and mainly etherified ferulic acids were also identified in the lignin fractions. It was found that the lignin fractions obtained from the two different grasses had very similar structural composition. They are distinguished by rather low amounts of β-O-4 structures, resinol units (β-β), and of condensed units (β-5 and 5-5). This trait is even more pronounced in the production of phenolic compounds for chemical industry from grass.     

Chemical composition and characterization of a galactomannoglucan from Gliocladium viride wall material

The following fractions were obtained from the wall material of Gliocladium viride : F1 (27.5%), a glucan, containing xylose, mannose and galactose, coluble in 1 M NaOH at 20°C; F2 (6.7%), a β-glucan-chitin complex, solubilized with 1 M NaOH at 20°C from the previous residue left overnight at −20°C; F3 (8.1%), a glucan, containing mannose and galactose solubilized with 1 M NaOH at 70°C; and F4, the insoluble residue, a β-glucan-chitin complex similar to F2, amounting to 31.3% of the wall material.
F1 was extracted with distilled water. The soluble material (F1S) was a galactomannoglucan (54.7%) and the inscluble (F1P) a glucan (45.3%). Periodate oxidation revealed the presence of glycerol, erythritol, threitol, ribitol, arabitol, mannose, galactose and glucose in F1S, and glycerol and glucose as the main components in F1P. The fractions obtained when F1S was purified through Sepharose CL6B, were methylated.     

β-Galactosidase from Lactobacillus pentosus: Purification,characterization and formation of galacto-oligosaccharides

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U_oNPG/mg protein. The K_m, k_cat and k_cat/K_m values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s^-1, 530 M^-1·s^-1 and 1.67 mM, 540 s^-1, 325 000 M^-1·s^-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg²⁺ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.     

Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan from Ganoderma lucidum

Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and ¹³C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight M_w and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The M_w value (2.4×10⁴) of sulfated glucan S-GL-1 was much lower than that (44.5×10⁴) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C_∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10^-3M_w^1.06 (cm³ g^-1) and 16, respectively, in the M_w range from 1.1×10⁴ to 2.4×10⁴. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Comparative studies on the polysaccharides of Cladonia alpestris (reindeer moss), Cladonia confusa,and Cladonia amaurocraea

The chemical structures of polysaccharide components of three species of the lichen genus Cladonia were compared. C. alpestris and C. confusa are similar in overall growth appearance despite different habitats, and each contains traces of water-insoluble nigeran. The residual lichens gave almost pure d-galacto-d-mannans isolated via insoluble Cu complexes formed with Fehling solution. They were not identical but structurally related having (1→6)-linked α-d-mannopyranosyl main-chains substituted in different patterns by β-d-galacto- and α-d-mannopyranosyl groups. Supernatant solutions of the Fehling-solution precipitation contained high proportions of β-d-galactofuranosyl residues. The polysaccharide of C. alpestris contained consecutive (1→2)-linked α-d-mannopyranosyl units substituted in the 6-position by β-d-galactofuranose, whereas that of C. confusa was a d-galactan with both pyranosyl and furanosyl forms. The d-glucan component of C. amaurocraea was isolated together with d-galacto-d-mannan as insoluble Cu complexes. The former was isolated in good yield and proved to be water-insoluble pustulan. The galactomannan had the same overall structure as those of C. alpestris and C. confusa, but showed differences according to the ¹³C-n.m.r. spectra.     

Structure of an l-arabino-d-xylan from the bark of Cinnamomum zeylanicum

An arabinoxylan isolated from the bark of Cinnamomum zeylanicum was composed of l-arabinose and d-xylose in the molar ratio 1.6:1.0. Partial hydrolysis furnished oligosaccharides which were characterised as α-d-Xylp-(1→3)-d-Ara, β-dXylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-β-d-Xylp-Xylp-(1→4)-d-Xyl, xylopentaose, and xylohexaose. Mild acid hydrolysis of the arabinoxylan gave a degraded polysaccharide consisting of l-arabinose (8%) and d-xyolse (92%). Methylation analysis indicated the degraded polysaccharide to be a linear (1→4)-linked d-xlan in which some xylopyranosyl residues were substituted at O-2 or O-3 with l-arabinofuranosyl groups. These data together with the results of methylation analysis and periodate oxidation of the arabinoxylan suggested that it contained a (1→4)-linked β-d-xylan backbone in which each xylopyranosyl residue was substituted both at O-2 and O-3 with l-arabinofuranosyl, 3-O-α-d-xylopyranosyl-l-arabinofuranosyl, and 3-O-l-arabinofuranosyl-l-arabinofuranosyl groups.     

Isolation and Structural Characterization of Lignin Polymer from Dendrocalamus sinicus

Dendrocalamus sinicus, which is the largest bamboo species in the world, has broad prospects in the fields of bioenergy and biorefinery application. In this study, dewaxed D. sinicus samples were sequentially treated with 80 % ethanol containing 0.025 M HCl, 80 % ethanol containing 0.5 % NaOH, and aqueous alkaline solutions (containing 2.0, 5.0, and 8.0 % NaOH, respectively) at 75 °C for 4 h, in which 9.63, 8.71, 21.83, 21.09, and 13.09 % of the original lignin were isolated, respectively. The lignin fractions obtained were comparatively characterized by chemical composition, molecular weights, and structural features by wet chemical and instrumental analysis methods. It was found that the bamboo lignin fractions isolated by ethanol had lower weight-average molecular weights (1,360–1,380 g?mol^?1) and contained much higher amounts of associated hemicelluloses, while the lignin fractions isolated by aqueous alkaline solutions had higher weight-average molecular weights (5,300–6,040 g?mol^?1) and contained lower amounts of associated hemicelluloses. Spectroscopy analyses indicated that the bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. A small percentage of the lignin side-chain was found to be acetylated at the γ-carbon, predominantly at syringyl units. The major interunit linkages present in the bamboo lignin obtained were β-O-4′ aryl ether linkages, together with lower amounts of β-β′, β-5′, and β-1′ linkages.     

The Uptake and Fractional Distribution of Differentially Labeled Indoleacetic Acid in Light Grown Stems

The uptake of exogenously applied indoleacetic acid (IAA) by light grown stems of bean (Phaseolus vulgaris L. cv. Red kidney) and pea (Pisum sativum L. cv. Alaska) was examined. The IAA was labeled in the 1 and 2 side chain positions with ¹⁴C and the 5 ring position with ³H. The distribution of label in the sections was analyzed by recording the elution into water, ethanol and 1.0 N NaOH, and the amount in the insoluble residue also recorded. Total uptake consisted of a rapid uptake for about 1 h followed by continued uptake at a slower rate for 24 h to give a radioactive concentration in the tissues four to five times, that of the external solution. Most of the radioactivity was initially extractable by water, later by ethanol. With IAA-2-¹⁴C there was a slow increase in radioactivity in NaOH and residue fractions but with IAA-1-¹⁴C most of the radioactivity was present in insoluble residue at times longer than 3 h. From the different residue patterns estimates of the extent of decarboxylation of the IAA were made. The radioactivity in the tissues was largely IAA after 1 h and the content increased until 6 h but there after there was little further increase. The water fraction initially contained the most IAA but by 24 h most IAA was found in the NaOH fraction in bean and the ethanol fraction in pea. The NaOH fraction was the only fraction in which the IAA content continually increased.     

Effects of temperature on carbon fixation and carbon budget partitioning in the zooxanthellal symbiosis of Aiptasia pallida (Verrill)

Effects of temperature on carbon fixation rates and partitioning between Aiptasia pallida (Verrill) and its symbiotic alga Symbiodinium microadriaticum Freudenthal were examined by ¹⁴C incubation studies. Total fixation varied strongly with temperature, with an optimum of 32 °C. More photosynthate was translocated to the host at 12 °C (82%) than at 27 °C (63%). Partitioning among three fractions (alcohol soluble, ether soluble, and alcohol/ether insoluble) varied with temperature in Aiptasia pallida, but not in the alga. Relative partitioning between host and alga increased with time in favor of A. pallida when maintained at 12 °C, but absolute levels of translocation to the host did not change; however, photosynthate retention by the alga did decline substantially. Total fixation declined by ≈ 80% after 10 days at 12 °C. Turnover rates of fixed carbon also varied with temperature, as determined by pulse-chase studies, and the effect varied for the different fractions.These results suggest that zooxanthellae are less thermally adaptable than their hosts, and may be especially susceptible to low temperatures. Thermal effects on biochemical partitioning may have great importance in relation to growth and reproduction of animal hosts of zooxanthellae and the viability of the symbiotic relationship. These effects, combined with the pronounced effect of temperature on total photosynthate production, probably play a major role in limitation of zooxanthellal symbioses to warm waters.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Comparative studies on beta-glucan hydrolases. Isolation and characterization of an exo(1 yields 3)-beta-glucanase from the snail, Helix pomatia.

An exo-β-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-200, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades β-(1 → 3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1→3; 1→4)-β-glucans are also extensively degraded and β-(1→6)- and β-(1→4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-β-glucanases, reported previously, in the broadness of its substrate specificity. The K_m values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50–55 °C.     

Biochemical characterization of a thermostable β-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K _m value was 10.35 mg/ml and the k _cat value was 588.24 s^?1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.     

The dependence of the conformation of a (1→3)-β-D-glucan on chain-length in alkaline solution

(1→3)-β-D-Glucans of various degrees of polymerization were prepared by degradation of a gel-forming D-glucan with formic acid. The degraded D-glucans were separated into a water-soluble fraction (soluble D-glucan) and an insoluble fraction (insoluble D-glucan). Both D-glucans were further fractionated. The optical rotation including determination of the o.r.d. curves of the fractions and of the original gel-forming D-glucans was measured at various sodium hydroxide concentrations (0–5M). The results indicate that (1→3)-β-D-glucans of $\text{DP}$n below ca. 25 (the soluble D-glucan) took a disordered form in both neutral and alkaline solutions, whereas the D-glucans of higher $\text{DP}$n (the insoluble and the original D-glucans) took an ordered structure in dilute alkaline solution (0.1M). The proportion of ordered structure in the insoluble D-glucan increases with $\text{DP}$n to attain a maximum value at a $\text{DP}$n of around 200; this may be the lower limit of $\text{DP}$n to permit gel formation in neutral media. The formation of complexes with Congo Red in alkaline solutions by the soluble and the insoluble D-glucans supports the same conclusions.     

Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization

Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni²⁺-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k _cat/K _m of 100 s^?1 μM^?1 for α-NA, and the value is 29.78 s^?1 μM^?1 for β-naphthyl acetate. The V _max and K _m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu²⁺ and Mg²⁺ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.