Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL^?1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL^?1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the ?3 and ?4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the ?4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL^?1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2?5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2?6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100?Ile732     

Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc₂Man₃-Dansyl was faster than that of Glc₁Man₃-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc₂Man₉GlcNAc₂ and Glc₁Man₉GlcNAc₂) although the activity was low.     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-β-glycosidase and β-fucosidase

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.     

Enzymatic synthesis of p-nitrophenyl 35-O-β-N-acetylglucosaminyl|-α-maltopentaoside by lysozyme; a novel substrate for human amylase assay

Transglycosylation from di-N-acetylchitobiose to the 3-position at the nonreducing end glucosyl group of p-nitrophenyl α-maltopentaoside was regioselectively induced through the use of hen egg-white lysozome. The enzyme formed p-nitrophenyl 3⁵-O-β-N-acetylglucosaminyl-α-maltopentaoside (5% of the enzyme-catalyzed net decreased of p-nitrophenyl α-maltopentaoside) from di-N-acetylchitobiose as a donor and p-nitrophenyl α-maltopentaoside as an acceptor. The rate of the transglycosylation depended on the concentration of substrate, the temperature and the pH. The hydrolytic actions of human pancreatic and salivary α-amylase on this derivative were examined. The maltopentaoside derivative was shown to be useful as a substrate for α-amylase assay through a coupled reaction involving α-D-glucosidase and glucoamylase.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.     

Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40°C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 ± 160 U mg⁻¹) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

The first α-1,3-glucosidase from bacterial origin belonging to glycoside hydrolase family 31

Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES–GroEL chaperones. Recombinant LJAG31 was an α-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k_cat/K_m) toward nigerose, maltulose, and kojibiose than other natural substrates having an α-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial α-1,3-glucosidase to be characterized with a high k_cat/K_m value for nigerose [α-d-Glcp-(1 → 3)-d-Glcp]. Transglucosylation of 4-nitrophenyl α-d-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl α-nigeroside and 4-nitrophenyl α-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) α-1,3-glucosidase of glycoside hydrolase family 31.     

Glycoside hydrolase family 31 Escherichia coli α-xylosidase

Escherichia coli YicI is a retaining α-xylosidase, which strictly recognizes the α-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli α-xylosidase (YicI). Detection of distinguishing features between α-xylosidases and α-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an α-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, α-d-Xylp-(1→6)-d-Manp, α-d-Xylp-(1→6)-d-Fruf, and α-d-Xylp-(1→3)-d-Frup, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal α-glucosidases.     

α-Galactosidase from Alfalfa Seeds (Medicago sativa L.)

An α-galactosidase from alfalfa seeds was purified 140-fold by ammonium sulfate fractionation, and column chromatography on Sephadex G-100, DEAE- and CM-Sephadex. Polyacrylamide-gel electrophoresis of the purified enzyme showed a single protein band. The molecular weight was estimated to be approximately 57,000 by gel-filtration. The purified enzyme hydrolyzed p-nitrophenyl α-d-galactoside more rapidly than raffinose. The maximal enzyme activities were obtained at pH 4.0 and 5.5 for p-nitrophenyl α-d-galactoside and at 4.5 for raffinose. The enzyme was shown to be inhibited by Hg²⁺ and Ag⁺ ions, and d-galactose.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Motif-Guided Identification of a Glycoside Hydrolase Family 1 α-L-Arabinofuranosidase in Bifidobacterium adolescentis

Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.     

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K _m and k _cat of 39 μM and 25.8 s^?1, respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80–114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.     

PURIFICATION AND PROPERTIES OF HUMAN BRAIN α-L-FUCOSIDASE

Human brain α-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity) were found in the purified α-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,000 ± 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 μmol/min/mg protein, respectively. Ag²⁺ and Hg²⁺ completely inactivated the enzyme at concentrations of 0.1-0.3 mM. Antibodies made previously against purified human liver α-L-fucosidase cross-reacted with the purified brain α-L-fucosidase and gave a single precipitin line coincident with that from purified liver α-L-fucosidase. From all our studies it appears that at least the soluble portion of brain α-L-fucosidase is identical to human liver α-L-fucosidase.     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.