Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.     

A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, β-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-β-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.     

Production and characterization of β-1,4-glucosidase from a strain of Penicillium pinophilum

A high β-glucosidase (BGL)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer rDNA gene sequence. Under the optimal culture conditions, a maximum BGL specific activity of 3.2 U ml⁻¹ (83 U mg-protein⁻¹), one of the highest levels among BGL-producing microorganisms was obtained. An extracellular BGL was purified to homogeneity by sequential chromatography of P. pinophilum culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a Mono Q column. The relative molecular weight of P. pinophilum BGL was determined to be 120 kDa by SDS-PAGE and size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 3.5 and a temperature optimum of 32 °C. P. pinophilum BGL showed a higher activity (V_max = 1120 U mg-protein⁻¹) than most BGLs purified from other sources. The internal amino acid sequences of P. pinophilum BGL showed a significant homology with hydrolases from glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, P. pinophilum BGL is distinguished from other BGLs by its high activity.     

A d-glucose- and d-xylose-tolerant GH1 β-glucosidase from Cellulosimicrobium funkei HY-13, a fibrolytic gut bacterium of Eisenia fetida

The GluM gene (1491-bp) coding for a β-glucosidase comprising a single catalytic glycoside hydrolase family 1 domain from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium funkei HY-13, was cloned and over-expressed in Escherichia coli BL21. The recombinant histidine-tagged enzyme (rGluM: 56 kDa) displayed the highest cleavage activity toward p-nitrophenyl (pNP)-β-d-glucopyranoside at pH 5.0 and 40 °C. The β-glucosidase activity of rGluM was enhanced over 1.8-fold of its original activity in the presence of 1 mM Ca²⁺, Ni²⁺, Mn²⁺, and Co²⁺ ions, respectively, while it was highly sensitive to 5 mM N-bromosuccinimide and 1 mM Hg²⁺. The susceptibility of some pNP-sugar derivatives and d-cellobiose to rGluM was evaluated to be in the order of pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > d-cellobiose > pNP-β-d-cellobioside > pNP-β-d-mannopyranoside. The k_cat/K_m values of rGluM toward pNP-β-d-glucopyranoside, pNP-β-d-galactopyranoside, and d-cellobiose were 302.28, 179.73, and 6.40 mM^-1 s^-1, respectively. At a concentration below 1.0 M, d-galactose was a potent activator of rGluM with β-glucosidase activity enhanced by approximately 160% in a dose-dependent manner. Moreover, the d-glucose (< 400 mM) and d-xylose (≤ 700 mM) stimulation of rGluM suggests that it can be exploited as a potential biocatalyst to generate d-glucose molecules in d-cellobiose degradation.     

Cloning and nucleotide sequence of β-mannanase and cellulase genes from Bacillus sp. 5H

The two genes for -mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the -mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M_r 40,803 Da (-mannanase) and 55,420 Da (cellulase). The deduced primary structure of -mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.     

Biocatalytic properties and substrate-binding ability of a modular GH10 β-1,4-xylanase from an insect-symbiotic bacterium,Streptomyces mexicanus HY-14

The gene (1350-bp) encoding a modular β-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and p-nitrophenyl-xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 β-1,4-xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction.     

Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K_m, and k_cat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg^-1, 0.23 mM, and 305 s^-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.     

Cloning, expression and characterization of a new ι-carrageenase from marine bacterium, Cellulophaga sp.

Purpose of work

The purpose of this study is to report a ι-carrageenase which degrades ι-carrageenan yielding neo-ι-carratetraose as the main product in the absence of NaCl. The gene for a new ι-carrageenase, CgiB_Ce, from Cellulophaga sp. QY3 was cloned and sequenced. It comprised an ORF of 1,386 bp encoding for a protein of 461 amino acid residues. From its sequence analysis, CgiB_Ce is a new member of GH family 82 and shared the highest identity of 32 % in amino acids with ι-carrageenase CgiA2 from Zobellia galactanovorans indicating that it is a hitherto uncharacterized protein. The recombinant CgiB_Ce had maximum specific activity (1,870 U/mg) at 45 °C and pH 6.5. It was stable between pH 6.0–9.6 and below 40 °C. Although its activity was enhanced by NaCl, the enzyme was active in the absence of NaCl. CgiB_Ce is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product (more than 80 % of the total product).     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

Improving the specific activity of β-mannanase from Aspergillus niger BK01 by structure-based rational design

β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57 Å crystal structure of ManBK. The protein adopts a typical (β/α)₈ fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18 ± 2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher k_cat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.     

Purification and characterization of a complex-bound and a free β-1,4-endoxylanase from the culture fluid of the anaerobic fungus Piromyces sp. strain E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50°C and the enzymes are stable up to 72h at 40°C. The enzymes have a pI of 9.1. The K _m and V _max, determined with xylan from oat spelts, were 3 mg · ml^-1 and 2600 IU · mg^-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl--d-glucoside, and p-nitrophenyl--d-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4--d-xylan xylanohydrolases, EC 3.2.1.8).     

Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium

An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum. Received: 19 March 1998 /  Received revision: 8 June 1998 / Accepted: 14 June 1998     

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

Marine bacterium Reinekea sp. KIT-YO10 was isolated from the seashore of Kanazawa Port in Japan as a seaweed-degrading bacterium. Homology between KIT-YO10 16S rDNA and the 16S rDNA of Reinekea blandensis and Reinekea marinisedimentorum was 96.4 and 95.4%, respectively. Endo-1,4-β-D-mannanase (β-mannanase, EC 3.2.1.78) from Reinekea sp. KIT-YO10 was purified 29.4-fold to a 21% yield using anion exchange chromatography. The purified enzyme had a molecular mass of 44.3?kDa, as estimated by SDS-PAGE. Furthermore, the purified enzyme displayed high specificity for konjac glucomannan, with no secondary agarase and arginase activity detected. Hydrolysis of konjac glucomannan and locust bean gum yielded oligosaccharides, compatible with an endo mode of substrate depolymerization. The purified enzyme possessed transglycosylation activity when mannooligosaccharides (mannotriose or mannotetraose) were used as substrates. Optimal pH and temperature were determined to be 8.0 and 70?°C, respectively. It showed thermostability at temperatures from 20 to 50?°C and alkaline stability up to pH 10.0. The current enzyme was thermostable and thermophile compared to the β-mannanase of other marine bacteria.     

Expression and characterization of GH3 β-Glucosidase from Aspergillus niger NL-1 with high specific activity, glucose inhibition and solvent tolerance

A β-glucosidase gene bglI from Aspergillus niger NL-1 was cloned and expressed in Pichia pastoris. The bglI gene consists of a 2583 bp open reading frame encoding 861 amino acids; the enzyme was classified into glycoside hydrolases 3. To improve the expression level of recombinant BGL in P. pastoris, fermentation conditions were optimized by the single-factor experiments. The optimal fermentation conditions were obtained: initial pH 5.0, methanol concentration 0.5% added into the culture every 24 h, and initial cell density (OD₆₀₀) of 10 for induction. The activity of BGL was increased from 4 U/mL to 45 U/mL in optimal conditions. The BGL was purified by ultrafiltration and (NH₄)₂SO₄ precipitation showing a single band on SDS-PAGE. The optimal activity was at pH 4.0 and 60°C. The recombinant enzyme was stable over a pH range of 3.0–7.0 and retained more than 85% activity after incubation at 60°C for 30 min. The kinetic experiments revealed K _m and V _max for p-nitrophenyl-β-D-glucoside of 0.64 mM and 370 U/mg, for cellobiose 8.59 mM and 1480 U/mg. The activity of BGL was not or only a little affected by many metal ions and EDTA and was enhanced by methanol or n-butyl alcohol. The BGL had a K _i of 48 mM for glucose and retained 76% activity in the presence of 50 mM glucose. The favorable properties of BGL offer the potential for industrial application.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

High level expression of a novel β-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis

A novel β-mannanase gene (CsMan5A) was cloned from Chaetomium sp. CQ31 and expressed in Pichia pastoris. It had an open reading frame of 1251 bp encoding 416 amino acids and contained two introns. The deduced amino acid sequence shared the highest similarity (73%) with the β-mannanase from Emericella nidulans and belongs to glycosyl hydrolase family 5. The recombinant β-mannanase (CsMan5A) was secreted at extremely high levels of 50,030 U mL⁻¹ and 6.1 mg mL⁻¹ in high cell density fermentor. The purified enzyme was optimally active at pH 5.0 and 65 °C and displayed broad pH stability (pH 5.0-11.0) and exhibited specificity towards locust bean gum (K_m = 3.1 mg mL⁻¹), guar gum (K_m = 9.3 mg mL⁻¹) and konjac powder (K_m = 10.5 mg mL⁻¹). It efficiently degraded mannan polysaccharides into mannose and mannooligosacccharides, and also hydrolyzed mannotriose and mannotetraose. These properties make CsMan5A highly useful in food, feed and paper/pulp industries.