Properties of γ-glutamyl arylamidase activity of the heavy subunit of γ-glutamyl arylamidase from Bacillus sp. strain No. 12

γ-Glutamyl arylamidase of Bacillus sp. strain No. 12, composed of two heavy (M_r 56 000) and two light (M_r 46 000) subunits, was dissociated and inactivated by mild SDS treatment. The activity was restored in the isolated heavy subunit but not in the light subunit when SDS was removed by dialysis. The restored activity of the heavy subunit was similar to that of the native enzyme with regard to substrate specificity and inhibition and activation by α- and γ-glutamyl compounds, free amino acids, peptides, enzyme inhibitors, and anti-native enzyme antibody.     

Purification and characterization of a β-cyclodextrin glucosyltransferase from an alkalophilic Bacillus sp.

Summary A -cyclodextrin glucosyltransferase was purified from alkalophilic Bacillus sp. No. 562 over 64-fold with a yield of 32%. Its molecular size was estimated to be 170 kDa by gel filtration and 82 kDa by SDS-PAGE, with a pI of 7.2. The enzyme showed optimum activity at 65 °C and pH 7.0. It was stable from 0 to70 °C and from pH 7.0 to 11.0. The enzyme was specifically inhibited by Fe2+ and Fe3+.     

Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.     

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     

Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase from Bacillus subtilis TAM-4

Two isozymes of γ-glutamyltranspeptidase, GGT-A and GGT-B, were purified to electrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(γ-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23,000 (I), 39,000 (II), and 40,000 (III). GGT-B was composed of two subunits with molecular weights of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55°C) but showed a significantly different thermal stability at 55°C. Both GGT-A and -B used d-γ-glutamyl-p-nitroanilide as well as the l-isomer as the γ-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.     

Purification and Properties of α-Glucosidase from Bacillus cereus

α-Glucosidase has been isolated from Bacillus cereus in ultracentrifugally and electrophoretically homogeneous form, and its properties have been investigated. The enzyme has a sedimentation constant of 1.4 S and a molecular weight of 12,000. The highly purified enzyme splits α-d-(1→4)-glucosidic linkages in maltose, maltotriose, and phenyl α-maltoside, but shows little or no activity toward polysaccharides, such as amylose, amylopectin, glycogen and soluble starch. The enzyme has α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme can also catalyze the transfer of α-glucosyl residue from maltose to riboflavin. On the basis of inhibition studies with diazonium-1-H-tetrazole, rose bengal and p-chloromercuribenzoate, it is assumed that the enzyme contains both histidine and cysteine residues in the active center.     

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.     

Purification and Some Properties of β-Glucosidase from Streptomyces sp

β-Glucosidases I, II, and III were isolated from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite column chromatography, filtration on Bio-Gel P-100, and DE-52 column chromatography. β-Glucosidase III had a single active band on disc-gel electrophoresis. Its optimum pH and temperature for activity were 6.0 and 60°C, respectively. The isoelectric point and molecular weight of the enzyme were pH 4.5 and 45,000, respectively. From an experiment using ¹⁴C-labeled glucose, gentiobiose seemed to be formed from laminaribiose as isomaltose is formed from maltose by fungal α-glucosidase. The enzyme showed transglucosylation and produced gentiobiose from β-gluco-disaccharides and 4-O-β-d-glucopyranosyl-d-manno-pyranose (epicellobiose). The enzyme acted on phenolic β-d-glucosides to produce unknown transfer products.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Purification and properties of 5′-nucleotidase from alkalophilic Bacillus No. C-3

5′-Nucleotidase (EC 3. 1. 3. 5) from alkalophilic Bacillus no. C-3 was purified to homogeneity. The molecular weight of the enzyme was 80,000 by gel filtration. The optimum pH for the activity was 9.5, and the enzyme was stable at pH 9.5–10.5 in a buffer containing 10 mM 2-mercaptoethanol. Substrate specificity study revealed that the enzyme acted on 5′-AMP strongly, on several 5′-nucleotides and ADP to a certain extent, but not on 3′-nucleotides, 2′-nucleotides, p-nitrophenyl phosphate, or ATP. The K_m value for 5′-AMP was 3.0 × 10⁻⁴ M. The enzyme required no divalent cation for its activity. The enzyme was inhibited by borate and arsenite ions but not by 1 mM EDTA.     

Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin (CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD.     

Synthesis of γ-Glutamyl Peptides Catalyzed by Transamidase from Bacillus natto

Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine. Both l- and d-isomers of glutamine were active as substrate. On incubation of l- or d-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed. The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides. The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with l-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from l-glutamine was a l-l isomer, while the dipeptide from d-glutamine was a d-d isomer.     

Purification and Properties of meso-α,∊-Diaminopimelate Decarboxylase from Bacillus sphaericus

Soybean 7S and 11S globulins were stored at relative humidities (RHs) of 11% and 96% at 50°C. The redispersibility of the proteins at RH 96% decreased in a short time. However, it did not decrease, when stored for 45 days at RH 11%. Gel filtration showed that the proteins polymerized during storage. The effects of urea, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on the redispersibilities of the proteins at RH 96% showed that the hydrogen, hydrophobic and disulfide bonds participate in the polymerization of 7S globulin, and that the disulfide bond is strongly related to the polymerization of 11S globulin. Redispersibility was restored with 2-ME in both the 7S and 11S globulins and some of the proteins in the supernatant redispersed with 2-ME were observed to be similar to the native ones with respect to the gel filtration, electrophoretic behavior and circular dichroism spectrum.     

Purification and Some Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.     

Purification,Crystallization and Some Enzymatic Properties of Acid Protease of Cladosporium sp. No. 45–2

An acid protease of Cladosporium sp. No. 45–2 was purified and crystallized by precipitation with ammonium sulfate, fractional precipitation with acetone, and pH adjustment. About 600 mg of third crystallized preparation was obtained from one liter of culture broth. The purified enzyme was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria such as uhracentrifugal and electrophoretical analysis. The enzyme was most active at pH values between 2.5 and 2.7 toward both casein and hemoglobin and was stable at pH values from 2.5 to 7.0 on twenty hour incubation at 30°C.

Millimolar concentration of sodium lauryl sulfate markedly inhibited the enzyme, wheares diisopropyl phosphorofluoridate, sulfhydryl reagents, ethylenediaminetetra acetic acid, and divalent metal ion relatively little affected the activity. The enzyme was most resistant toward S-PI among the acid proteases tested.     

Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp.

The endo α-1,4 polygalactosaminidase from Pseudomonas sp. 881 was purified from the culture nitrate by ethanol precipitation and sequential column chromatographies on CM-Sephadex C-25, Sephadex G-50 and Phenyl-Sepharose CL-4B. The purified enzyme was electrophoretically homogeneous and its molecular weight and isoelectric point were 31,000 and 6.7, respectively. The optimum pH and temperature for hydrolysis of polygalactosamine were 5.0 and 55°C, respectively. The enzyme was stable up to 45°C for 15min and from pH 4.0 to 7.6 at 37°C for 1 hr.

The Km value was 0.05% α-1,4 polygalactosamine and the V was 0.154μmol reducing sugar (galactosamine)/min/μg protein. This polygalactosaminidase was inhibited by Sn²⁺ , Fe²⁺ , Fe³⁺ , Hg²⁺, Cu²⁺ ions and SDS. The enzyme did not hydrolyze oligo galactosamines (n < tetramer) or N-acetyl-polygalactosamines. It acted only on oligo galactosamine (n > trimer) and polygalactosamine endogeneously so far tested.     

Purification and Properties of NADP-Dependent Maltose Dehydrogenase Produced by Alkalophilic Corynebacterium sp. No. 93–1

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93–1. The molecular weight of the enzyme was estimated as 45,000~48,000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1 mm NADP. The enzyme oxidized d-xylose, maltose and maltotriose, and the Km values for these substrates were 150mm, 250 mm and 270 mm, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5 mm with 100mm maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-δ-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.     

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic Thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.