Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

A kinetic correlation for konjac powder hydrolysis by β-mannanase from Bacillus licheniformis

The kinetics of konjac powder hydrolysis by -mannanase from Bacillus licheniformis was studied for the production of manno-oligosaccharides, a bifidobacteria factor. It is assumed that, during the whole reaction, the substrate has an average degree of polymerization with which the apparent kinetic constants, K _m and k ₊₂, vary and the concentration of substrate remains unchanged. Based on the above assumptions, a set of simple expressions is proposed to correlate the initial concentration of substrate, enzyme concentration, degree of hydrolysis and reaction time. The results predicted are in good agreement with the experimental data.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Improving the acidic stability of a β-mannanase from Bacillus subtilis by site-directed mutagenesis

β-Mannanase can randomly hydrolyze the (1→4)-β-d-mannosidic linkages in mannans, galactomannans and glucomannans, yielding manno-oligosaccharides. In this study, the β-mannanase (MAN) from Bacillus subtilis B10-02 was overexpressed successfully in B. subtilis 168 as a hexa-histidine tagged, secreted protein. The recombinant enzyme BsMAN6H was not stable under acidic conditions, which restricts its use in food and feed industry. We aimed to improve the acid stability of BsMAN6H by changing several surface-exposed amino acid residues to acidic or neutral ones. Among the mutations, the His54Asp resulted in a shift in the optimal pH from 6.5 to 5.5. Accordingly, the acid stability was improved by a factor of a negative potential on the structure surface around the mutated site. Furthermore, the H54D variant showed the enzyme activity up to 3207.82 U/mL in bioreactors using the cheap Kojac powder as substrate. As a result, a bacterial β-mannanase was produced efficiently with increased acid stability, improving its applicability in the animal feed industry.     

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.     

Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Purification and Properties of β-Galactosidases from Bacillus circulans

Six compounds, Z- and E-fadyenolide (3, 4), 1-ally1-2,3-(methylenedioxy)-4,5-dimethoxy-benzene (5), 4-methoxy-3,5-bis (3′-methyl-2′-butenyl)-benzoic acid (6), 2,6-dihydroxy-4-methoxy-dihydrochalcone (7), and 5-hydroxy-7-methoxyflavanone (8) were isolated from three species of Jamaican Piper, Piper fadyenii, C.D.C., Piper aduncum L. and Piper hispidum Sw. Three amides (9 ~ 11) of 3,5-dimethoxy-4-oxo-5-phenylpent-2-enoic acid using piperidine, pyrrolidine and morpholine, respectively, were synthesized from compounds 3 and 4, and tested for insecticidal activity against the tick Boophilus microplus (Canestrini) and the flour feetle, Tribolium confusum Duval. In our experiment, compounds 9 ~ 11 inhibited ovogenesis of B. microplus and were toxic to T. confusum. Compounds 3 ~ 8 were found to have no activity.     

Cloning, sequence analysis, and heterologous expression of a β-mannanase gene from Bacillus subtilis Z-2

Mannanase, an extracellular enzyme that catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has potential to be applied in food industries. In this study a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with a deduced molecular mass of 38 kDa. The man gene can both be expressed heterologously into the periplasm from the plasmid pET22b(+) containing an intact signal peptide (pET-NdeI18) or the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. The E. coli DH5α expression of man was under the control of the lac promoter in the pRK415 vector; it was much more effective when the Shine Dalgarno (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for a high-level mannanase expression in different bacterial strains. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 418–424. This article was submitted by the authors in English.     

Purification of α-amylase from Bacillus licheniformis by chromatofocusing and gel filtration chromatography

A combination of chromatofocusing and gel filtration chromatography resulted in a simple purification of -amylase from Bacillus licheniformis. The purification was approximately 77-fold. Identification of the purity was established by SDS–PAGE. Molecular weight and isoelectric point of the purified enzyme were 58 kDa and 7.18 respectively. Western blot analysis confirms the specificity of antibody raised against purified -amylase.     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

Cloning, Functional Expression and Characterization of an Alkaline Protease from Bacillus licheniformis

A gene (apr 46) encoding a protease was cloned from Bacillus licheniformis RSP-09-37. It had an ORF of 1725 bp, encoding a pre-protein of 575 amino acids (63.2 kDa), which was functionally expressed and processed in E. coli JM 109. The mature protein, Apr 46, consists of 500 amino acids with a calculated molecular mass of 55 kDa. This protease shows 29-50% homology to known serine proteases and conserved domains. N-terminal sequencing suggests that Apr 46 protease is identical to a B. licheniformis RSP-09-37 protease, which is further supported by a similar stability in acetonitrile.     

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.     

Purification and properties of β-amylase from Bacillus circulans S31

A thermotolerant -amylase was purified from Bacillus circulans S31 isolated from soil in Hong Kong. The purified enzyme has an M _r of 64 kDa and was stable at 50°C and pH 7.0 for 30 min. Its K _m for starch was 0.9 mg/ml with a V _max of 0.3 mg/min. It was not activated by any metal ion although sulphydrys reagents were inhibitory.H.S. Kwan, K.H. So and K.Y. Chan are with the Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong S.C. Cheng is with the Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic, Hung Hom, Hong Kong.     

Hemicellulolytic and cellulolytic functions of the domains of a β-mannanase cloned from Caldicellosiruptor saccharolyticus

Various combinations of the four domains of the multifunctional mannanase from Caldicellosiruptor saccharolyticus have been cloned and expressed in Escherichia coli. The four domains comprise two catalytic domains (1 and 4), and two putative cellulose binding domains (2 and 3). Each of the six gene products (Man1, Man123, Man1234, Man23, Man234 and Man4) was partially purified by heat treatment.The enzymes Man1234, Man123 and Man1 exhibited activity on mannans, and Man1234, Man234 and Man4 exhibited activity on xylan and carboxymethylcellulose (CMC). For the complete enzyme (Man1234) all activities were of the same order of magnitude. Activities were additive against a mixture of mannan and xylan or mannan and CMC (but not xylan and CMC). The expression product Man23 exhibited activity on none of the substrates tested, nor did its presence influence thermostability or significantly reduce the K_m value for any of the substrates. However, when expressed in combination with domains 1 or 4 it greatly increased their activity.We conclude that domain 1 catalyses mannan hydrolysis and domain 4 catalyses xylan and CMC hydrolysis at the same active site: domains 2 and 3 have no obvious function, since they do not reduce substrate K_m nor affect thermostability. However, their effect on rates of substrate hydrolysis may indicate a role influencing the conformation of the adjacent catalytic domains.     

Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis

Summary An extracellular -glucanase from Bacillus licheniformis has been isolated and characterized. Isolation has been performed by salting out and gel filtration chromatography, yielding a homogeneous active component with a molecular mass of 27 000–28 000 daltons and an isoelectric point of 4.7. In addition to being quite a thermostable protein (optimal temperature 55°C) the enzyme is active under a wide range of conditions including pH (4.0–10.5), and in the presence of a large number of metal ions, sodium dodecylsulphate and ethylenediaminetetraacetate. The simple purification procedure and useful properties make this enzyme suitable for brewing processes.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Purification and some properties of β-mannanase fromPolyporus versicolor

Multiple enzyme forms of -mannanase activity fromPolyporus versicolor were puritied to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel filtration on Sephadex G-100 and high-performance liquid chromatography using anion exchange and hydrophobic Interaction media. Overall, 7.6% of input activity was recovered in four -mannanases, A, B, C and 2A, which were purified 112.6-, 165.5-, 143.7-and 19.9-fold respectivety. The -mannanases were acidic proteins displaying isoelectric points from 3.75 to 4.6, molecular weights in the range of 33,900 to 57,500 and increasing hydrophobicity in the order of C>B>2A>A. Optimal pH and temperature for the hydrotysis of glucomannan by all activities were pH 5.5 and 65°C, respectively. All preparations exhibited activity after 30 min at 65°C, or after protease digestion. Although the response of individual enzymes to selected ions was variable, all -mannanases were inhibited in decreasing order of Hg²⁺>Cu²⁺>Zn²⁺>Mn²⁺. All activities functioned as endomannanases.

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Résumé De multiples formes enzymatiques de l'activité -mannanasique dePolyporus verslcolor ont été purifiées jusqu'à l'homogénéité moléculaire par une séquence impliquant la chromatographie sur Bio-gel DEAE A, la filtration sur gel de Sephadex G-100, et la chromatographie liquide à haute performance utilisant l'échange anionique et les milieux à interaction hydrophobique. On a récupéré en tout 7.6% de l'activité Initiale dans quatre -mannanases, A, B, C, et 2A, qui ont été purifiées respectivement 112.6, 165.5, 143.7 et 19.9 fois. Les -mannanases sont des protéines acidiques exhibant des pointsiso-électriques de 3.75 à 4.6, des poids moléculaires compris entre 33 900 et 57 500, et une hydrophobicité croîssante dans l'ordre C>B>2A>A. Les pH et température optimum pour l'hydrolyse de la glucamannane par toutes les activités sont de 5.5 et 65°C respectivement. Toutes les préparations exhibent encore une activité après 30 minutes et 65°C ou après la digestion protéolytique. Bien que la réponse individuelle des enzymes à quelques ions choisis était variable, toutes les -mannanases sont inhibées dans l'ordre décroissant: Hg²⁺>Cu²⁺>Zn²⁺>Mn²⁺. Toutes les activités fonctionnent comme endomannanases.

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This article is issued as NRCC No. 31269.